Conversion Of Α-ketoglutarate Research Articles

Abstract 2180: Deciphering the Warburg effect: Redox is the key to tumor differentiation

Abstract It was proposed by Warburg that the cause and consequence of the Warburg effect were the injury of respiration and cell dedifferentiation, respectively. However, the underlying mechanism was unclear due to limited understanding of metabolic control of epigenetics. Inhibition of mitochondrial electron transport chain (ETC) causes reduction of NAD+/NADH ratio. Recently it was discovered that this redox stress not only drives lactate production from pyruvate to enhance the Warburg effect, but also promotes the conversion of α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), turning the substrate of DNA demethylase TET into an inhibitor of TET, suggesting a potential link between ETC inhibition and dysregulated DNA methylation in cancer. In this study we use a mitochondrial uncoupler niclosamide ethanolamine (NEN) to block the Warburg effect and remodel cancer epigenome. NEN treatment activates ETC and accelerates glutaminolysis to produce αKG, which promotes promoter CpG island demethylation. This epigenomic reprograming not only upregulates tumor suppressor genes such as p53, inhibits N-Myc expression, but also activates neuron differentiation markers to promote differentiation in neuroblastoma cells. Hypoxia is one common microenvironmental stress existing in all solid tumor. NEN treatment reduces 2HG generation from αKG under hypoxia, highlighting its application potential in preventing DNA hypermethylation in vivo. Associated with 2HG reduction, NEN treatment also represses HIF signaling under hypoxia. In addition, NEN treatment inhibits reductive carboxylation flux. Moreover, dietary supplementation with NEN reduces orthotopic neuroblastoma growth and N-Myc expression in vivo. Together, these results validated Warburg’s hypothesis that mitochondrial respiration is the key to maintain normal differentiation. Restoring ETC activity with mitochondrial uncoupler is an effective metabolic/epigenetic therapy against neuroblastoma (Jiang et al., BioRxiv 2021). Citation Format: Jiang Haowen, Rachel L. Greathouse, Bo He, Yang Li, Albert M. LI, Balint Forgo, Selene Banuelos, Joshua Gruber, Hiroyuki Shimada, Bill Chiu, Jiangbin Ye. Deciphering the Warburg effect: Redox is the key to tumor differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2180.

Discovery of Five New Ethylene-Forming Enzymes for Clean Production of Ethylene in E. coli.

Ethylene is an essential platform chemical with a conjugated double bond, which can produce many secondary chemical products through copolymerisation. At present, ethylene production is mainly from petroleum fractionation and cracking, which are unsustainable in the long term, and harmful to our environment. Therefore, a hot research field is seeking a cleaner method for ethylene production. Based on the model ethylene-forming enzyme (Efe) AAD16440.1 (6vp4.1.A) from Pseudomonas syringae pv. phaseolicol, we evaluated five putative Efe protein sequences using the data derived from phylogenetic analyses and the conservation of their catalytic structures. Then, pBAD expression frameworks were constructed, and relevant enzymes were expressed in E. coli BL21. Finally, enzymatic activity in vitro and in vivo was detected to demonstrate their catalytic activity. Our results show that the activity in vitro measured by the conversion of α-ketoglutarate was from 0.21–0.72 μmol ethylene/mg/min, which varied across the temperatures. In cells, the activity of the new Efes was 12.28–147.43 μmol/gDCW/h (DCW, dry cellular weight). Both results prove that all the five putative Efes could produce ethylene.

A Theoretical Model of Mitochondrial ATP Synthase Deficiencies. The Role of Mitochondrial Carriers

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (neuropathy, ataxia and retinitis pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-c-oxidase, preventing yeast growth on oxidative substrates. The overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) was able to rescue the growth on the oxidative substrate by increasing the substrate-level phosphorylation of ADP coupled to the conversion of α-ketoglutarate (AKG) into succinate with an increase in Complex IV activity. Previous studies showed that equivalent point mutations in ATP synthase behave similarly and can be rescued by Odc1p overexpression and/or the uncoupling of OXPHOS from ATP synthesis. In order to better understand the mechanism of the ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as a respiratory substrate. We describe the different possible metabolite outputs and the ATP/O ratio values as a function of ATP synthase inhibition.

Metabolic changes related to the IDH1 mutation in gliomas preserve TCA-cycle activity: An investigation at the protein level.

The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA-cycle activity in IDH1 mut gliomas.

An olive oil phenolic is a new chemotype of mutant isocitrate dehydrogenase 1 (IDH1) inhibitors.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene confer an oncogenic gain-of-function activity that allows the conversion of α-ketoglutarate (α-KG) to the oncometabolite R-2-hydroxyglutarate (2HG). The accumulation of 2HG inhibits α-KG-dependent histone and DNA demethylases, thereby generating genome-wide hypermethylation phenotypes with cancer-initiating properties. Several chemotypes of mutant IDH1/2-targeted inhibitors have been reported, and some of them are under evaluation in clinical trials. However, the recognition of acquired resistance to such inhibitors within a few years of clinical use raises an urgent need to discover new mutant IDH1 antagonists. Here, we report that a naturally occurring phenolic compound in extra-virgin olive oil (EVOO) selectively inhibits the production of 2HG by neomorphic IDH1 mutations. In silico docking, molecular dynamics, including steered simulations, predicted the ability of the oleoside decarboxymethyl oleuropein aglycone (DOA) to preferentially occupy the allosteric pocket of mutant IDH1. DOA inhibited the enzymatic activity of recombinant mutant IDH1 (R132H) protein in the low micromolar range, whereas >10-fold higher concentrations were required to inhibit the activity of wild-type (WT) IDH1. DOA suppressed 2HG overproduction in engineered human cells expressing a heterozygous IDH1-R132H mutation. DOA restored the 2HG-suppressed activity of histone demethylases as it fully reversed the hypermethylation of H3K9me3 in IDH1-mutant cells. DOA epigenetically restored the expression of PD-L1, an immunosuppressive gene silenced in IDH1 mutant cells via 2HG-driven DNA hypermethylation. DOA selectively blocked colony formation of IDH1 mutant cells while sparing WT IDH1 isogenic counterparts. In sum, the EVOO-derived oleoside DOA is a new, naturally occurring chemotype of mutant IDH1 inhibitors.

Elucidation of a Self-Sustaining Cycle in Escherichia coli l-Serine Biosynthesis That Results in the Conservation of the Coenzyme, NAD.

The equilibrium of the reaction catalyzed by d-3-phosphoglycerate dehydrogenase (PGDH), the first enzyme in the l-serine biosynthetic pathway, is far in the direction away from serine synthesis. As such, the enzyme is usually assayed in this direction. To easily assay it in the direction of l-serine synthesis, it can be coupled to the next enzyme in the pathway, phosphoserine aminotransferase (PSAT), with the activity monitored by the conversion of NAD+ to NADH by PGDH. However, when PGDHs from several different species were coupled to PSAT, it was found that one of them, ecPGDH, conserves the coenzyme in the production of l-serine by utilizing an intrinsic cycle of NAD+/NADH interconversion coupled with the conversion of α-ketoglutarate (αKG) to α-hydroxyglutarate. Furthermore, the cycle can be maintained by production of αKG by the second enzyme in the pathway, PSAT, and does not require any additional enzymes. This is not the case for PGDH from another bacterial source, Mycobacterium tuberculosis, and a mammalian source, human liver, where net consumption of NAD+ occurs. Both NAD+ and NADH appear to remain tightly bound to ecPGDH during the cycle, effectively removing a requirement for the presence of an exogenous coenzyme pool to maintain the pathway and significantly reducing the energy requirement needed to maintain this major metabolic pathway.

Tissue-specific metabolism and TRIM24

Epigenetic regulation of metabolism is critical to maintain cellular homeostasis in response to cellular demands and resources. Changes in the microenvironment impact functions of epigenetic regulatory enzymes and metabolic balance must maintained to avoid metabolic reprogramming and tumor progression [1]. Epigenetic regulatory proteins with catalytic functions include histone “writers” or “erasers”, which effect post-translational modifications (PTMs) of histones or non-histone proteins; chromatin remodelers, fueled by ATP to alter chromatin structure; and, enzymes that methylate DNA or reverse DNA methylation. Genes encoding key factors in metabolic signaling pathways are regulated by the activities of these enzymes, which remodel or modify chromatin. Additionally, epigenetic regulatory enzymes themselves serve as environmental sensors by relying on available metabolites as cofactors. For example, mutations in IDH1/IDH2, which encode isocitrate dehydrogenases of the tricarboxylic acid (TCA) cycle, are associated with low-grade gliomas, adult de novo acute myeloid leukemias and lymphomas. Mutant IDH1/2 exhibit gain-of-function by catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxy glutarate (2-HG) at 100-fold higher levels than found in normal cells. 2-HG is a competitive inhibitor of α-KG-dependent dioxygenases, including TET2, which acts in reversal of DNA methylation, and Jumonji-C-domain-containing histone demethylases, which alter histone PTMS; either of which may disrupt regulated gene expression [2]. In addition to epigenetic regulatory enzymes that act as sensors of cellular metabolites and/or directly alter chromatin structure of genes that encode metabolic enzymes, proteins known as “histone readers” serve as relay switches in regulatory networks, including metabolism. Histone readers have specific domains that bind defined “signatures” of histone PTMs and act as platforms for recruitment of transcription factors, mediators or additional epigenetic response factors to chromatin. Our laboratory showed that histone reader Tripartite motif-containing protein 24 (TRIM24) not only negatively regulates p53 as an E3-ubiquitin ligase [3] but also interacts with and recruits transcription factors, such as estrogen receptor, to chromatin via a tandem plant homeodomain (PHD) and Bromodomain that binds unmethylated lysine 4 and acetylated lysine 23 of histone H3 (H3K4me0/H3K23ac) [4]. More recently, we found that ectopic expression of TRIM24 promoted oncogenic transformation of immortalized human mammary epithelial cells (TRIM24-iHMECs) and efficient growth of intermediate to high-grade xenograft tumors [5]. Molecular analysis of TRIM24-iHMECs revealed a TRIM24-dependent glycolytic and TCA cycle gene expression signature, which led to increased glucose uptake. Gene Set Enrichment Analysis revealed the glucose transport pathway as one of the top 10 pathways positively correlated with TRIM24 expression in human breast tumors (n = 1008) from the TCGA database. Interestingly, Seahorse analysis showed both ECAR (measure of glycolysis) and OCR (measure of OXPHOS) were elevated in TRIM24-iHMECs. This is unlike a conventional Warburg effect of unrestrained, aerobic glycolysis but consistent with recent reports of cancers that exploit OXPHOS or a mixture of glycolysis and OXPHOS for energy production [6]. In addition to its functions as a histone reader, TRIM24 is an E3-ubiquitin ligase that targets p53 for protein degradation. Tumor suppressor p53 plays key roles in glycolysis, OXPHOS, glutamine metabolism, lipid metabolism and antioxidant defense to impact cellular metabolism and redox balance [7]. We surveyed both p53-positive and -negative breast cancer-derived cell lines to assess p53-dependence of TRIM24-regulated metabolic response and found that GLUT1, ACO1, IDH1 and IDH2, which encode important players in glycolysis and TCA cycle, were TRIM24-activated in MCF7, SKBR3 and MDAMB231 cells despite their varied p53 status. This may be explained by our finding that TRIM24 directly regulates these genes, as well as another important player in oncogenesis and metabolism: c-myc. Our unpublished chromatin immunoprecipitation analyses show direct binding of TRIM24 to the upstream regulatory regions of c-myc, GLUT1, IDH1 and IDH2, concomitant with activation of transcription. Given the clinical correlates in breast cancer patients and apparent oncogenic effects on metabolism that we reported in human mammary epithelial cells, one might expect that loss of TRIM24 in mouse models would oppose tumor development. However, complete loss of Trim24, either globally or conditionally in the liver, caused spontaneous hepatic steatosis and ultimately hepatocellular carcinoma in animals fed normal chow. With loss of Trim24 expression, hepatocytes increased expression of lipase and inflammation signaling genes and repressed de novo lipogenesis, steroid and lipid metabolism and transport. The hepatic accumulation of lipids, fibrosis and infiltration of inflammatory macrophages recapitulated parameters of human nonalcoholic fatty liver disease (NAFLD) and non-obese-NASH [8]. Whether this outcome is the result of tissuespecific shifts in transcription factors that collaborate with TRIM24, an altered collection of TRIM24 target genes and/or a tissue-specific response to regulated p53 levels is unknown at this time. Clearly, additional mouse models, especially those with conditional over expression of Trim24, and manipulation of diet, along with mechanistic studies of functional domains and intersecting pathways, are needed to determine how epigenetic variables and tissue-specificity dictate metabolic reprogramming by TRIM24.

Abstract 2438: Defining the role of mutant isocitrate dehydrogenase in malignant glioma

Abstract Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) were recently found in ∼80% of WHO grade II-IV gliomas. These mutations inhibit the enzyme's ability to convert isocitrate to α-ketoglutarate and, instead, confer a novel gain-of-function resulting in the conversion of α-ketoglutarate to 2-hydroxglutarate. However, the fundamental mechanism(s) by which these mutations affect glioma cell growth remain unclear. IDH1 and 2 function as homodimers in the cytosol and mitochondria, respectively. Dimerization is mediated by interactions between the two clasp domains of each IDH protein and dimeric IDH contains two active sites each composed of amino acid residues from both subunits. Thus, the dimerization of IDH is vital for its enzymatic role within the cell. Interestingly, IDH mutations have been observed as heterozygous events in glioma cells suggesting that the cells require the wild-type IDH protein in order to function. To examine and visualize the interaction between wild-type and mutant IDH proteins, we employed the protein-protein interaction assay: Bimolecular Fluorescence Complementation (BiFC). This approach is based on the formation of a fluorescent complex though the association of two fragments of a fluorescent reporter protein fused to two proteins of interest. An interaction between the proteins facilitates association between the two fragments to produce a bimolecular fluorescent complex. Since IDH1 has a C-terminal peroxisomal targeting sequence, the N-terminal 172 amino acid residues of Venus (VN) were fused to the N-terminus of wild-type IDH1 and residues 155-238 amino acids of Venus (VC) were fused to the N-terminus of mutant IDH1. IDH2, however, has an N-terminal mitochondrial targeting sequence and thus the N-terminal and C-terminal fragments of Venus were fused to the C-terminus of wild-type and mutant IDH2, respectively. 293FT cells were transfected with each fluorescent reporter construct and counterstained with nuclear and cytosolic markers to confirm complex formation and cellular localization. Approximately 60% of the cells showed evidence of IDH complex formation. We confirmed localization to the cytosol/peroxisomes and mitochondria for wild-type and mutant IDH1 and IDH2 heterodimers, respectively, and observed that, in the case of IDH1, wild-type and mutant heterodimers demonstrate physiologically normal activity compared to inactive mutant IDH1 homodimers. Conversely, we observed no significant difference between wild-type and mutant IDH2 heterodimers when compared to mutant IDH2 homodimers. This data suggests that mutant IDH1 requires wild-type IDH1 in order to be enzymatically active while mutant IDH2 does not require wild-type IDH2. To determine the effect of wildtype and mutant IDH heterodimer formation on glioma cell growth, we plan to assess the tumorigenic potential of IDH1 heterodimers either alone or in concert with other known glioma genetic abnormalities in vivo. Citation Format: Gemma L. Robinson, Matthew R. Guthrie, Marytheresa Ifediba, Matthew W. VanBrocklin, Sheri L. Holmen. Defining the role of mutant isocitrate dehydrogenase in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2014-2438

Inhibition of δ-Aminolevulinic Acid Synthesis by Glyphosate

The effect of glyphosate [N-(phosphonomethyl) glycine] on barley (Hordeum vulgareL.) and corn (Zea maysL.) shoot δ-aminolevulinic acid (ALA) production was examined by monitoring ALA content in the tissue and measuring incorporation of14C precursors into ALA and chlorophylla. Barley shoot ALA content was significantly decreased by 1 mM glyphosate after 9, 11, and 15 h of illumination. ALA production by treated barley shoots was 30 nmoles•g fresh weight-1•h-1at each interval tested, compared with 75 to 120 nmoles•g fresh weight-1•h-1for the control. In corn shoots, ALA content was reduced 32, 45, and 58% by 0.1, 1.0, and 10.0 mM glyphosate, respectively, after 12 h illumination. Incorporation studies with14C-glutamate,14C-α-ketoglutarate, and14C-glycine into ALA showed a 77, 92, and 91% inhibition, respectively, in barley shoots treated with 1 mM glyphosate. Incorporation of14C-ALA into chlorophyllawas not affected by 1 mM glyphosate. Thus, the site of action of glyphosate may involve two enzyme pathways:one controlling the conversion of α-ketoglutarate to ALA, and the other controlling the condensation of glycine with succinyl CoA to form ALA and carbon dioxide. Inhibition of ALA synthesis blocks synthesis of chlorophyll, as well as all other porphyrin ring compounds found in higher plants. Thus, inhibition of ALA synthesis may be an integral component of the herbicidal mode of action of glyphosate.

Lack of Citrate Lyase — the Key Enzyme of the Reductive Carboxylic Acid Cycle — in Chlorobium thiosulfatophilum and Rhodospirillum rubrum

Extracts of Chlorobium thiosulfatophilum and of Rhodospirillum rubrum have been tested for the presence of citrate lyase under various conditions. This enzyme could not be detected. It is, therefore, concluded that a complete reductive carboxylic acid cycle does not occur in these microorganisms. The enzymes required for a-ketoglutarate synthesis from oxaloaetate and acetyl-coenzyme A are present in autotrophically grown cells of C. thiosulfatophilum and R. rubrum. This supports the view that a reaction sequence catalyzing the conversion of α-ketoglutarate into oxaloacetate is unlikely to exist in these bacteria.