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An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.

Succination is the spontaneous reaction between the respiratory intermediate fumarate and cellular thiols that forms stable S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC). 2SC is a biomarker for conditions associated with elevated fumarate levels, including diabetes, obesity, and certain cancers, and succination likely contributes to disease progression. Bacillus subtilis has a yxe operon-encoded breakdown pathway for 2SC that involves three distinct enzymatic conversions. The first step is N-acetylation of 2SC by YxeL to form N-acetyl-2SC (2SNAC). YxeK catalyzes the oxygenation of 2SNAC, resulting in its breakdown to oxaloacetate and N-acetylcysteine, which is deacetylated by YxeP to give cysteine. The monooxygenase YxeK is key to the pathway but is rare, with close homologs occurring infrequently in prokaryote and fungal genomes. The existence of additional 2SC breakdown pathways was not known prior to this study. Here, we used comparative genomics to identify a S-(2-succino) lyase (2SL) that replaces yxeK in some yxe gene clusters. 2SL genes from Enterococcus italicus and Dickeya dadantii complement B. subtilis yxeK mutants. We also determined that recombinant 2SL enzymes efficiently break down 2SNAC into fumarate and N-acetylcysteine, can perform the reverse reaction, and have minor activity against 2SC and other small molecule thiols. The strong preferences both YxeK and 2SL enzymes have for 2SNAC indicate that 2SC acetylation is a conserved breakdown step. The identification of a second naturally occurring 2SC breakdown pathway underscores the importance of 2SC catabolism and defines a general strategy for 2SC breakdown involving acetylation, breakdown, and deacetylation.

In the glucose-rich milieu of red blood cells, asexually replicating malarial parasites mainly rely on glycolysis for ATP production, with limited carbon flux through the mitochondrial tricarboxylic acid (TCA) cycle. By contrast, gametocytes and mosquito-stage parasites exhibit an increased dependence on the TCA cycle and oxidative phosphorylation for more economical energy generation. Prior genetic studies supported these stage-specific metabolic preferences by revealing that six of eight TCA cycle enzymes are completely dispensable during the asexual blood stages of Plasmodium falciparum, with only fumarate hydratase (FH) and malate-quinone oxidoreductase (MQO) being refractory to deletion. Several hypotheses have been put forth to explain the possible essentiality of FH and MQO, including their participation in a malate shuttle between the mitochondrial matrix and the cytosol. However, using newer genetic techniques like CRISPR and dimerizable Cre, we were able to generate deletion strains of FH and MQO in P. falciparum. We employed metabolomic analyses to characterize a double knockout mutant of FH and MQO (ΔFM) and identified changes in purine salvage and urea cycle metabolism that may help to limit fumarate accumulation. Correspondingly, we found that the ΔFM mutant was more sensitive to exogenous fumarate, which is known to cause toxicity by modifying and inactivating proteins and metabolites. Overall, our data indicate that P. falciparum is able to adequately compensate for the loss of FH and MQO, rendering them unsuitable targets for drug development.

Cellular thiols such as cysteine spontaneously and readily react with the respiratory intermediate fumarate, resulting in the formation of stable S-(2-succino)-adducts. Fumarate-mediated succination of thiols increases in certain tumors and in response to glucotoxicity associated with diabetes. Therefore, S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC) are considered oncometabolites and biomarkers for human disease. No disposal routes for S-(2-succino)-compounds have been reported prior to this study. Here, we show that Bacillus subtilis metabolizes 2SC to cysteine using a pathway encoded by the yxe operon. The first step is N-acetylation of 2SC followed by an oxygenation that we propose results in the release of oxaloacetate and N-acetylcysteine, which is deacetylated to give cysteine. Knockouts of the genes predicted to mediate each step in the pathway lose the ability to grow on 2SC as the sulfur source and accumulate the expected upstream metabolite(s). We further show that N-acetylation of 2SC relieves toxicity. This is the first demonstration of a metabolic disposal route for any S-(2-succino)-compound, paving the way toward the identification of corresponding pathways in other species.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882–9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf. The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe(2+) and 2-oxoglutarate-binding residues increased the Km values for these factors 30-80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400-500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.

Inactivating mutations of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH) have been linked to an aggressive variant of hereditary kidney cancer (hereditary leiomyomatosis and renal cell cancer). These tumors accumulate markedly elevated levels of fumarate. Fumarate is among a growing list of oncometabolites identified in cancers with mutations of genes involved in intermediary metabolism. FH-deficient tumors are notable for their pronounced accumulation of the transcription factor hypoxia inducible factor-1α (HIF-1α) and aggressive behavior. To date, HIF-1α accumulation in hereditary leiomyomatosis and renal cell cancer tumors is thought to result from fumarate-dependent inhibition of prolyl hydroxylases and subsequent evasion from von Hippel-Lindau-dependent degradation. Here, we demonstrate a novel mechanism by which fumarate promotes HIF-1α mRNA and protein accumulation independent of the von Hippel-Lindau pathway. Here we demonstrate that fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank binding kinase 1 (TBK1). Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1α accumulation in cellular models of FH loss and markedly reduces cell invasion of FH-deficient RCC cancer cells. Collectively, our data demonstrate a novel mechanism by which pseudohypoxia is promoted in FH-deficient tumors and identifies TBK1 as a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors.

Dimethyl fumarate (DMF) is an effective novel treatment for multiple sclerosis in clinical trials. A reduction of IFN-γ-producing CD4(+) T cells is observed in DMF-treated patients and may contribute to its clinical efficacy. However, the cellular and molecular mechanisms behind this clinical observation are unclear. In this study, we investigated the effects of DMF on dendritic cell (DC) maturation and subsequent DC-mediated T cell responses. We show that DMF inhibits DC maturation by reducing inflammatory cytokine production (IL-12 and IL-6) and the expression of MHC class II, CD80, and CD86. Importantly, this immature DC phenotype generated fewer activated T cells that were characterized by decreased IFN-γ and IL-17 production. Further molecular studies demonstrated that DMF impaired nuclear factor κB (NF-κB) signaling via reduced p65 nuclear translocalization and phosphorylation. NF-κB signaling was further decreased by DMF-mediated suppression of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its downstream kinase mitogen stress-activated kinase 1 (MSK1). MSK1 suppression resulted in decreased p65 phosphorylation at serine 276 and reduced histone phosphorylation at serine 10. As a consequence, DMF appears to reduce p65 transcriptional activity both directly and indirectly by promoting a silent chromatin environment. Finally, treatment of DCs with the MSK1 inhibitor H89 partially mimicked the effects of DMF on the DC signaling pathway and impaired DC maturation. Taken together, these studies indicate that by suppression of both NF-κB and ERK1/2-MSK1 signaling, DMF inhibits maturation of DCs and subsequently Th1 and Th17 cell differentiation.

Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is upregulated more than 20-fold under anaerobic conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_OA_fulltext

Mechanistic details of mammalian metabolism in vivo and dynamic metabolic changes in intact organisms are difficult to monitor because of the lack of spatial, chemical, or temporal resolution when applying traditional analytical tools. These limitations can be addressed by sensitivity enhancement technology for fast in vivo NMR assays of enzymatic fluxes in tissues of interest. We apply this methodology to characterize organ-specific short chain fatty acid metabolism and the changes of carnitine and coenzyme A pools in ischemia reperfusion. This is achieved by assaying acetyl-CoA synthetase and acetyl-carnitine transferase catalyzed transformations in vivo. The fast and predominant flux of acetate and propionate signal into acyl-carnitine pools shows the efficient buffering of free CoA levels. Sizeable acetyl-carnitine formation from exogenous acetate is even found in liver, where acetyl-CoA synthetase and acetyl-carnitine transferase activities have been assumed sequestered in different compartments. In vivo assays of altered acetate metabolism were applied to characterize pathological changes of acetate metabolism upon ischemia. Coenzyme pools in ischemic skeletal muscle are reduced in vivo even 1 h after disturbing muscle perfusion. Impaired mitochondrial metabolism and slow restoration of free CoA are corroborated by assays employing fumarate to show persistently reduced tricarboxylic acid (TCA) cycle activity upon ischemia. In the same animal model, anaerobic metabolism of pyruvate and tissue perfusion normalize faster than mitochondrial bioenergetics.

DcuB of Escherichia coli catalyzes C4-dicarboxylate/succinate antiport during growth by fumarate respiration. The expression of genes of fumarate respiration, including the genes for DcuB (dcuB) and fumarate reductase (frdABCD) is transcriptionally activated by C4-dicarboxylates via the DcuS-DcuR two-component system, comprising the sensor kinase DcuS, which contains a periplasmic sensing domain for C4-dicarboxylates. Deletion or inactivation of dcuB caused constitutive expression of DcuS-regulated genes in the absence of C4-dicarboxylates. The effect was specific for DcuB and not observed after inactivation of the homologous DcuA or the more distantly related DcuC transporter. Random and site-directed mutation identified three point mutations (T394I, D398N, and K353A) in DcuB that caused a similar derepression as dcuB deletion, whereas the transport activity of the DcuB mutants was retained. Constitutive expression in the dcuB mutants depended on the presence of a functional DcuS-DcuR two-component system. Mutation of residues E79A, R83A, and R127A of DcuB, on the other hand, inactivated growth by fumarate respiration and transport of [14C]succinate, whereas the expression of dcuB'-'lacZ was not affected. Therefore, the antiporter DcuB is a bifunctional protein and has a regulatory function that is independent from transport, and sites for transport and regulation can be differentiated.

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.

DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C(4) -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C(4)-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mm for the C(4)-dicarboxylates and 0.5 mm for 3-nitropropionate or d-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X(2)-His(110)-X(9)-Phe(120)-X(26)-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or d-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C(4)-dicarboylate binding domain. The amino acid residues responding to fumarate or d-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C(4)-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.

The two-component regulatory system DcuSR of Escherichia coli controls the expression of genes of C(4)-dicarboxylate metabolism in response to extracellular C(4)- dicarboxylates such as fumarate or succinate. DcuS is a membrane-integral sensor kinase, and the sensory and kinase domains are located on opposite sides of the cytoplasmic membrane. The intact DcuS protein (His(6)-DcuS) was overproduced and isolated in detergent containing buffer. His(6)-DcuS was reconstituted into liposomes made from E. coli phospholipids. Reconstituted His(6)-DcuS catalyzed, in contrast to the detergent-solubilized sensor, autophosphorylation by [gamma-(33)P]ATP with an approximate K(D) of 0.16 mm for ATP. Up to 7% of the reconstituted DcuS was phosphorylated. Phosphorylation was stimulated up to 5.9-fold by C(4)-dicarboxylates, but not by other carboxylates. The phosphoryl group of DcuS was rapidly transferred to the response regulator DcuR. Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA. The reconstituted DcuSR system therefore represents a defined in vitro system, which is capable of the complete transmembrane signal transduction by the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane.

Equilibration of maleate in D2O with a partially purified preparation of maleate hydratase from rabbit kidney resulted in the formation of d-malate containing 1 atom of stably bound deuterium per molecule. The enzyme therefore catalyzes a stereospecific addition to the double bond of maleate. The product of the enzymatic hydration in D2O was examined by nuclear magnetic resonance spectroscopy. In addition, the monodeuteriosuccinate obtained by chemical reduction of the deuterated d-malate was analyzed for its optical rotatory properties. The absolute configuration of the enzymatically deuterated malate was thus established and corresponded to the structure d-malate-3-d-(3-R). It was concluded that the over-all reaction catalyzed by maleate hydratase is trans.