Fumarate Hydratase Activity Research Articles

Identification of a Difluorinated Alkoxy Sulfonyl Chloride as a Novel Antitumor Agent for Hepatocellular Carcinoma through Activating Fumarate Hydratase Activity.

Fenofibrate is known as a lipid-lowering drug. Although previous studies have reported that fenofibrate exhibits potential antitumor activities, IC50 values of fenofibrate could be as high as 200 μM. Therefore, we investigated the antitumor activities of six synthesized fenofibrate derivatives. We discovered that one compound, SIOC-XJC-SF02, showed significant antiproliferative activity on human hepatocellular carcinoma (HCC) HCCLM3 cells and HepG2 cells (the IC50 values were 4.011 μM and 10.908 μM, respectively). We also found this compound could inhibit the migration of human HCC cells. Transmission electron microscope and flow cytometry assays demonstrated that this compound could induce apoptosis of human HCC cells. The potential binding sites of this compound acting on human HCC cells were identified by mass spectrometry-cellular thermal shift assay (MS-CETSA). Molecular docking, Western blot, and enzyme activity assay-validated binding sites in human HCC cells. The results showed that fumarate hydratase may be a potential binding site of this compound, exerting antitumor effects. A xenograft model in nude mice demonstrated the anti-liver cancer activity and the mechanism of action of this compound. These findings indicated that the antitumor effect of this compound may act via activating fumarate hydratase, and this compound may be a promising antitumor candidate for further investigation.

Integrated genome-centric metagenomic and metaproteomic analyses unravel the responses of the microbial community to ammonia stress

Ammonia is a major inhibitor in anaerobic digestion of nitrogen-rich organic wastes. In this study, integrated genome-centric metagenomic and metaproteomic analyses were used to identify the key microorganisms and metabolic links causing instability by characterizing the process performance, microbial community, and metabolic responses of key microorganisms during endogenous ammonia accumulation. The identification of 89 metagenome-assembled genomes and analysis of their abundance profile in different operational phases permitted the identification of key taxa (Firmicutes and Proteobacteria) causing poor performance. Metabolic reconstruction indicated that the key taxa had the genetic potential to participate in the metabolism of C2C5 volatile fatty acids (VFAs). Further investigation suggested that during Phase I, the total ammonia nitrogen (TAN) level was maintained below 2000 mg N/L, and the reactor showed a high methane yield (478.30 ± 33.35 mL/g VS) and low VFAs concentration. When the TAN accumulated to > 2000 mg N/L, acid accumulation, mainly of acetate, began to occur, and the methane yield gradually decreased to 330.44 mL/g VS (Phase II). During this phase, the VFA degradation functions of the community were mainly mediated by Firmicutes. Approximately 61.54% of significant differentially expressed proteins (DEPs) related to acetate metabolism in Firmicutes were down-regulated, which led to an increase in acetate concentration to 4897.91 ± 1558.96 mg/L. However, the reactor performance showed spontaneous recovery without any interference (Phase III), during which Firmicutes gradually adapted to the high ammonia conditions. Approximately 75% of the significant DEPs related to acetate metabolism of Proteobacteria were also up-regulated in Phase III compared with Phase II; thus, VFA-related metabolic functions of the community were enhanced, which resulted in a decrease in the total VFA concentration to 195.39 mg/L. When the TAN increased above 4000 mg N/L, the system gradually showed acid accumulation dominated by propionate, accompanied by a second decrease in methane yield (Phase IV). During this phase, the number of up-regulated and down-regulated proteins related to acetate metabolism of Firmicutes and butyrate/valerate metabolism of Proteobacteria was comparable with that of Phase III, indicating that the metabolic functions related to acetate, butyrate, and valerate of the microbial community were not significantly affected. However, for propionate metabolism, the expression activity of fumarate hydratase from Firmicutes and Proteobacteria was severely inhibited by ammonia, as shown by down-regulation ratios of 63.64% and 85.71%, respectively. No protein with the same function that was not inhibited by ammonia could be detected, and the fumarate degradation function of the microbial community was severely damaged, leading to blocked propionate metabolism and irreversible deterioration of reactor performance. This study has provided a new perspective on the microecological mechanisms of ammonia inhibition.

Retraction Note: Suppression of fumarate hydratase activity increases the efficacy of cisplatin-mediated chemotherapy in gastric cancer

Retraction Note: Suppression of fumarate hydratase activity increases the efficacy of cisplatin-mediated chemotherapy in gastric cancer

Abstract 3112: Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models

Abstract Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare but highly aggressive malignancies that are often refractory to the therapies used for the more common RCCs. FH-deficient RCC most often occurs in individuals with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) and is characterized by loss of FH activity, a key metabolic enzyme. Bevacizumab plus erlotinib (B+E) is a preferred first-line therapy for FH-deficient RCC. RMC is characterized by loss of SMARCB1, which is involved in ATP-dependent chromatin remodeling. Platinum-based chemotherapy consisting of either cisplatin or carboplatin plus paclitaxel is the preferred first-line therapy for RMC. We developed patient-derived xenograft (PDX) models of tumors exposed to the commonly used first-line therapies to allow functional characterization of the metabolic hallmarks of treatment-experienced RMC and FH-deficient RCC. Methods: Resected tumor tissue was immediately implanted into NSG male and female mice. After tumor engraftment, animals were euthanized, and the tumor tissue implanted into another set of NSG mice. PDX tissues were confirmed to be genetically identical to the original patient tissue by short tandem repeat analysis. Histology was confirmed to be identical between original patient tumor and PDX by a trained clinical pathologist. Four tumors from each PDX model and four normal human kidney tissue samples were analyzed using reverse phase protein array (RPPA). Expression of proteins shown to be highly expressed by RPPA were further validated by Western blots. Results: We successfully generated one PDX model for RMC and one for FH-deficient RCC, respectively. The RMC model was generated from a 28-year old male with sickle cell trait, who had previously received cisplatin plus paclitaxel followed carboplatin plus paclitaxel. His lesion was characterized as ypT3apN1pMx and histology was consistent with RMC. The FH-deficient RCC model was generated from a 24-year-old female with HLRCC, who had been previously treated with B+E. Her lesion was characterized as pT3apN1pM1 and histology consistent with FH-deficient RCC. Germline testing revealed a R233H pathogenic mutation in the FH gene. RPPA revealed higher expression of hexokinase II (10x HLRCC, 16x RMC), lactate dehydrogenase A (27x HLRCC, 17x RMC), and glutaminase (4x HLRCC) in PDX tissue compared to normal kidney tissue. Western blots confirmed higher expression of glutaminase 1 in both HLRCC and RMC compared to normal kidney. Furthermore, we observed substantially higher hypoxia inducible factor 2α (HIF2α) protein expression in PDX tissue from FH-deficient RCC compared to RMC PDX tumors and normal kidney. Conclusions: Our study revealed distinct metabolic features in PDX models of FH-deficient RCC following treatment with B+E, and of platinum-experienced RMC, these differences may confer targetable vulnerabilities. Citation Format: Manuel Ozambela, Alberto Pieretti, Graciela M. Nogueras Gonzalez, Tapati Maity, Lei Wang, Carolyn De La Cerda, Breanna Alonzo, Luis Segarra, Angelita Alaniz, Priya Rao, Nizar Tannir, Jose A. Karam, Christopher G. Wood, Pavlos Msaouel, Niki M. Zacharias. Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models [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 3112.

Nitrogen uptake efficiency induced fumarate hydratase activity in oil palm seedlings

Nitrogen uptake efficiency induced fumarate hydratase activity in oil palm seedlings

Correction: Chromatin Remodeling Factor LSH Drives Cancer Progression by Suppressing the Activity of Fumarate Hydratase

In the original version of this article (1), there was a duplication of the images used in migration assays for C666–1 cells treated with siLSH#1 and siLSH#2 in Fig. 3B. The error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.

The influence of high frequency electromagnetic field on the growth of lactic acid bacteria used in the food industry

The objects of the study were lactic acid bacteria Lactobacillus lactis and Lactobacillus casei used in the food industry. It is proposed to use an extremely high frequency electromagnetic field (EMF EHF) with a wavelength of 4.9 mm for 5 minutes to activate the metabolism of lactic acid bacteria. The stimulating effect of EMF EHF on the growth of biomass and excretion of lactic acid during the growth was found. Physiological constants characterizing the development of microorganisms have been determined. It was found that the maximum yield from the consumed substrate Ymax increased by 40% and the maximum specific growth rate rmax by 70% when exposed to radiation, the consumption rate for maintenance ms decreased by 10% and the substrate saturation constant Ks by 23%. A change in the activity of key enzymes of metabolic pathways of lactose utilization immediately after the action of EMF of the EHF was revealed. The preservation of the achieved effect during the entire fermentation was observed. The activity of β-galactosidase and hexokinase after exposure to EMF EHF increases from 134% to 290%, depending on the strain and fermentation conditions. The increase in the activity of fumarate hydratase is 6-9%.

Direct and quantitative analysis of altered metabolic flux distributions and cellular ATP production pathway in fumarate hydratase-diminished cells

Fumarate hydratase (FH) is an enzyme in the tricarboxylic acid (TCA) cycle, biallelic loss-of-function mutations of which are associated with hereditary leiomyomatosis and renal cell cancer. However, how FH defect modulates intracellular metabolic fluxes in human cells has remained unclear. This study aimed to reveal metabolic flux alterations induced by reduced FH activity. We applied 13C metabolic flux analysis (13C-MFA) to an established cell line with diminished FH activity (FHdim) and parental HEK293 cells. FHdim cells showed reduced pyruvate import flux into mitochondria and subsequent TCA cycle fluxes. Interestingly, the diminished FH activity decreased FH flux only by about 20%, suggesting a very low need for FH to maintain the oxidative TCA cycle. Cellular ATP production from the TCA cycle was dominantly suppressed compared with that from glycolysis in FHdim cells. Consistently, FHdim cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FHdim cells we demonstrated that FH defect led to suppressed pyruvate import into mitochondria, followed by downregulated TCA cycle activity and altered ATP production pathway balance from the TCA cycle to glycolysis. We confirmed that 13C-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect.

Hyperpolarized [1,4-13C]fumarate imaging detects microvascular complications and hypoxia mediated cell death in diabetic nephropathy

Today, there is a general lack of prognostic biomarkers for development of renal disease and in particular diabetic nephropathy. Increased glycolytic activity, lactate accumulation and altered mitochondrial oxygen utilization are hallmarks of diabetic kidney disease. Fumarate hydratase activity has been linked to mitochondrial dysfunction as well as activation of the hypoxia inducible factor, induction of apoptosis and necrosis. Here, we investigate fumarate hydratase activity in biofluids in combination with the molecular imaging probe, hyperpolarized [1,4-13C2]fumarate, to identify the early changes associated with hemodynamics and cell death in a streptozotocin rat model of type 1 diabetes. We found a significantly altered hemodynamic signature of [1,4-13C2]fumarate in the diabetic kidneys as well as an systemic increased metabolic conversion of fumarate-to-malate, indicative of increased cell death associated with progression of diabetes, while little to no renal specific conversion was observed. This suggest apoptosis as the main cause of cell death in the diabetic kidney. This is likely resulting from an increased reactive oxygen species production following uncoupling of the electron transport chain at complex II. The mechanism coupling the enzyme leakage and apoptotic phenotype is hypoxia inducible factor independent and seemingly functions as a protective mechanism in the kidney cells.

Tricarboxylic acid cycle enzyme activities in a mouse model of methylmalonic aciduria

Methylmalonic acidemia (MMA) is a propionate pathway disorder caused by dysfunction of the mitochondrial enzyme methylmalonyl-CoA mutase (MMUT). MMUT catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, an anaplerotic reaction which feeds into the tricarboxylic acid (TCA) cycle. As part of the pathological mechanisms of MMA, previous studies have suggested there is decreased TCA activity due to a “toxic inhibition” of TCA cycle enzymes by MMA related metabolites, in addition to reduced anaplerosis. Here, we have utilized mitochondria isolated from livers of a mouse model of MMA (Mut-ko/ki) and their littermate controls (Ki/wt) to examine the amounts and enzyme functions of most of the TCA cycle enzymes. We have performed mRNA quantification, protein semi-quantitation, and enzyme activity quantification for TCA cycle enzymes in these samples. Expression profiling showed increased mRNA levels of fumarate hydratase in the Mut-ko/ki samples, which by contrast had reduced protein levels as detected by immunoblot, while all other mRNA levels were unaltered. Immunoblotting also revealed decreased protein levels of 2-oxoglutarate dehydrogenase and malate dehydrogenase 2. Interesting, the decreased protein amount of 2-oxoglutarate dehydrogenase was reflected in decreased activity for this enzyme while there is a trend towards decreased activity of fumarate hydratase and malate dehydrogenase 2. Citrate synthase, isocitrate dehydrogenase 2/3, succinyl-CoA synthase, and succinate dehydrogenase are not statistically different in terms of quantity of enzyme or activity. Finally, we found decreased activity when examining the function of methylmalonyl-CoA mutase in series with succinate synthase and succinate dehydrogenase in the Mut-ko/ki mice compared to their littermate controls, as expected. This study demonstrates decreased activity of certain TCA cycle enzymes and by corollary decreased TCA cycle function, but it supports decreased protein quantity rather than “toxic inhibition” as the underlying mechanism of action. SummaryMethylmalonic acidemia (MMA) is an inborn metabolic disorder of propionate catabolism. In this disorder, toxic metabolites are considered to be the major pathogenic mechanism for acute and long-term complications. However, despite optimized therapies aimed at reducing metabolite levels, patients continue to suffer from late complications, including metabolic stroke and renal insufficiency. Since the propionate pathway feeds into the tricarboxylic acid (TCA) cycle, we investigated TCA cycle function in a constitutive MMA mouse model. We demonstrated decreased amounts of the TCA enzymes, Mdh2 and Ogdh as semi-quantified by immunoblot. Enzymatic activity of Ogdh is also decreased in the MMA mouse model compared to controls. Thus, when the enzyme amounts are decreased, we see the enzymatic activity also decreased to a similar extent for Ogdh. Further studies to elucidate the structural and/or functional links between the TCA cycle and propionate pathways might lead to new treatment approaches for MMA patients.

Abstract 2760: Photoinducible detection of the oncometabolite fumarate

Abstract Dysregulated metabolism is an important marker of many disease states, including cancer. For example, in hereditary leiomyomatosis and renal cell cancer (HLRCC), inactivating mutations in fumarate hydratase (FH) lead to accumulation of high levels of fumarate, a so-called “oncometabolite.” Substantial evidence indicates that fumarate stimulates various oncogenic signaling pathways, necessitating sensitive methods to detect the oncometabolite in order to more rapidly diagnose HLRCC as well as to identify new disease settings in which fumarate may play a signaling role. Here, we report development of novel photoactivatable, fluorogenic chemical probes for detection and profiling of fumarate in biological systems. These chemical probes, diaryl tetrazoles, are by themselves inert towards fumarate. However, upon irradiation with UV light, they release nitrileimines that can form fluorescent cycloadducts with fumarate. We have demonstrated that diaryl tetrazoles can sensitively detect FH activity as well as low micromolar levels of fumarate in complex biological samples. We have also shown that diaryl tetrazoles can be used to monitor changes in intracellular fumarate levels in biological samples by live-cell imaging and flow cytometry. Moreover, these compounds are capable of visualizing differences between patient-derived primary HLRCC tumors lacking FH activity and the adjacent normal kidney, highlighting their potential utility in clinical diagnostics. By offering new insights into fumarate reactivity, our studies provide the chemical basis for novel approaches to therapy and diagnosis in cancers driven by oncometabolite accumulation. Citation Format: Chloe Briney, Sarah Bergholtz, Rhushikesh Kulkarni, Daniel Crooks, Chandrasekhar Mushti, Stephen Lockett, Rolf Swenson, W. Marston Linehan, Jordan Meier. Photoinducible detection of the oncometabolite fumarate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2760.

RETRACTED ARTICLE: Suppression of fumarate hydratase activity increases the efficacy of cisplatin-mediated chemotherapy in gastric cancer

Gastric cancer (GC) is one of the most common malignancies worldwide. Due to the low rate of early detection, most GC patients were diagnosed as advance stages and had poor response to chemotherapy. Some studies found that Fumarate hydratase (FH) participated in the DNA damage response and its deficiency was associated with tumorigenesis in some cancers. In this study, we investigated the relationship between FH and cisplatin (CDDP) sensitivity in GC cell lines. We found that FH was the most significant gene which induced by CDDP treatment and the suppression of FH could enhance the cytotoxicity of CDDP. Miconazole Nitrate (MN) could inhibit FH activity and enhance the effect of CDDP in vitro and in vivo. We also investigated the significance of expression of FH in GC tissues. The FH expression, which was higher in GC tissues than in noncancerous tissues, was negatively associated with the prognosis of patients. Together, these results revealed that FH is a reliable indicator for response to CDDP treatment in GC and the inhibition of FH may be a potential strategy to improve the effects of CDDP-based chemotherapy.

Bioorthogonal oncometabolite ligation.

Dysregulated cellular metabolism is an emerging hallmark of cancer. Improved methods to profile aberrant metabolic activity thus have substantial applications as tools for diagnosis and understanding the biology of malignant tumors. Here we describe the utilization of a bioorthogonal ligation to fluorescently detect the TCA cycle oncometabolite fumarate. This method enables the facile measurement of fumarate hydratase activity in cell and tissue samples, and can be used to detect disruptions in metabolism that underlie the genetic cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The current method has substantial utility for sensitive fumarate hydratase activity profiling, and also provides a foundation for future applications in diagnostic detection and imaging of cancer metabolism.

Suppression of experimental cerebral malaria by disruption of malate:quinone oxidoreductase

BackgroundAspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is thus indispensable for the growth of Plasmodium parasites at the asexual blood stages. OAA can be produced in malaria parasites via two routes: (i) from phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol, or (ii) from fumarate by consecutive reactions catalyzed by fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO) in the mitochondria of malaria parasites. Although PEPC-deficient Plasmodium falciparum and Plasmodium berghei (rodent malaria) parasites show a growth defect, the mutant P. berghei can still cause experimental cerebral malaria (ECM) with similar dynamics to wild-type parasites. In contrast, the importance of FH and MQO for parasite viability, growth and virulence is not fully understood because no FH- and MQO-deficient P. falciparum has been established. In this study, the role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined.ResultsFirst, FH- and MQO-deficient parasites were generated by inserting a luciferase-expressing cassette into the fh and mqo loci in the genome of P. berghei ANKA strain. Second, the viability of FH-deficient and MQO-deficient parasites that express luciferase was determined by measuring luciferase activity, and the effect of FH or MQO deficiency on the development of ECM was examined. While the viability of FH-deficient P. berghei was comparable to that of control parasites, MQO-deficient parasites exhibited considerably reduced viability. FH activity derived from erythrocytes was also detected. This result and the absence of phenotype in FH-deficient P. berghei parasites suggest that fumarate can be metabolized to malate by host or parasite FH in P. berghei-infected erythrocytes. Furthermore, although the growth of FH- and MQO-deficient parasites was impaired, the development of ECM was suppressed only in mice infected with MQO-deficient parasites.ConclusionsThese findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.

Chromatin Remodeling Factor LSH Drives Cancer Progression by Suppressing the Activity of Fumarate Hydratase.

Chromatin modification is pivotal to the epithelial-mesenchymal transition (EMT), which confers potent metastatic potential to cancer cells. Here, we report a role for the chromatin remodeling factor lymphoid-specific helicase (LSH) in nasopharyngeal carcinoma (NPC), a prevalent cancer in China. LSH expression was increased in NPC, where it was controlled by the Epstein-Barr virus-encoded protein LMP1. In NPC cells in vitro and in vivo, LSH promoted cancer progression in part by regulating expression of fumarate hydratase (FH), a core component of the tricarboxylic acid cycle. LSH bound to the FH promoter, recruiting the epigenetic silencer factor G9a to repress FH transcription. Clinically, we found that the concentration of TCA intermediates in NPC patient sera was deregulated in the presence of LSH. RNAi-mediated silencing of FH mimicked LSH overexpression, establishing FH as downstream mediator of LSH effects. The TCA intermediates α-KG and citrate potentiated the malignant character of NPC cells, in part by altering IKKα-dependent EMT gene expression. In this manner, LSH furthered malignant progression of NPC by modifying cancer cell metabolism to support EMT. Cancer Res; 76(19); 5743-55. ©2016 AACR.

Chromatin remodeling factor LSH affects fumarate hydratase as a cancer driver.

Cancer metabolism and epigenetic alteration are two critical mechanisms for tumorigenesis and cancer progression; however, the dynamic interplay between them remains poorly understood. As reported in the article entitled “Chromatin remodeling factor LSH drives cancer progression by suppressing the activity of fumarate hydratase,” which was recently published in Cancer Research, our group examined the physiological role of lymphocyte-specific helicase (LSH) in nasopharyngeal carcinoma (NPC) by focusing on cancer progression and the tricarboxylic acid cycle. We found that LSH was overexpressed in NPC, and its expression associated with Epstein-Barr virus infection. We also found that LSH directly suppressed fumarate hydratase (FH), a key component of the tricarboxylic acid cycle, in combination with euchromatic histone-lysine N-methyltransferase 2 (EHMT2), also known as G9a. Depletion of FH promoted epithelial-mesenchymal transition (EMT). Moreover, LSH controlled expression of tricarboxylic acid cycle intermediates that promote cancer progression, including EMT, through activation by inhibitor of nuclear factor kappa-B kinase alpha (IKKα), a chromatin modifier and transcriptional activator. Our study showed that LSH plays a critical role in cancer progression, which has important implications for the development of novel strategies to treat NPC.

Phytochrome-dependent regulation of fumarate hydratase activity in maize green leaves

In green maize (Zea mays L.) leaves, a dependence of the activity of fumarate hydratase (FH) on light conditions was revealed. It was established that the phytochrome system is involved in the photo-regulation of the activity of this enzyme, and red light (RL) retarded the rate of its functioning. Investigation of the level of fum 1 and fum 2 gene expression in green leaves showed that only gene fum 1 is actively expressed, whereas expression of fum 2 gene is blocked. The data obtained provide evidence that calcium ions in maize leaves are capable of acting as a second messenger in the light signal transduction, and the active phytochrome form being formed under RL action caused an accumulation of calcium ions behaving as a regulator of Krebs cycle functioning. Employment of ruthenium red, a known inhibitor of cell membrane Ca2+-channels, and Ca2+ chelator, EGTA, allowed us to find that the RL-induced change in the content of free calcium ions in cell nuclei of maize leaves is associated with their redistribution between cell compartments. The active phytochrome form caused the increase in the rate of pif3 gene transcription that was accompanied by inhibiting the functioning of fum 1 gene. Based on these results, it is suggested that transcription factor PIF3 can mediate the transduction of the phytochrome signal in the nucleus.

The Succinated Proteome of FH-Mutant Tumours.

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC.

Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas

Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an 'SDH-like' molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2-succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P = 0.007) and multiple tumors (P = 0.02) were significantly more frequent in patients with FH mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior.

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP+ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.