Why is succinate oxidized by fad

In addition, two comprehensive comparison studies of tumours with SDHB or SDHD mutations have been performed and several obvious differences have been identified Neumann et al. It is noteworthy that SDHD mutations are mostly related to head and neck paraganglioma whereas SDHB mutations can be seen at higher frequencies in adrenal and extra-adrenal phaeochromocytoma as well as in non-paraganglioma tumours. SDHB mutations are also associated with higher incidences of malignant and metastatic tumours such as malignant phaeochromocytoma and, in some cases, renal cell carcinoma Vanharanta et al.

In , the FH gene at 1q43 was independently identified as the fourth mitochondrial tumour suppressor gene in families with the hereditary leiomyomatosis and renal cell carcinoma HLRCC syndrome Tomlinson et al.

Like the SDH genes, FH mutations lead to loss of function and are associated with loss of the wild-type allele and FH activity in the tumours Tomlinson et al.

It is important to state that the different types of tumours that develop from the dysfunction of either SDH or FH probably indicate that different mechanisms contribute variably to tumour phenotype.

Such mechanisms may also cause the small but significant genotype—phenotype differences observed between the SDH genes. Yet pseudo-hypoxia, the major phenomenon shown to date to mediate the tumorigenic outcome of the loss of mitochondrial tumour suppressors, is a common mechanism for both SDH and FH mutations.

Still, the existence of a common mechanism is in keeping with the fact that SDH and FH catalyse successive reactions of the TCA cycle and thus share a common pathway Figure 1. The link between the loss of mitochondria tumour suppressors and pseudo-hypoxia was indicated by several studies. Initial research by Gimenez-Roqueplo et al. This study explained the high vascular density observed in these paragangliomas.

Interestingly, in the past year, it was shown that FH-deficient tumours also display high vascularity Pollard et al. These independent studies collectively suggested that pseudo-hypoxia, manifested by high HIF activity under normoxic conditions, is an important factor that links mitochondrial tumour suppressors and cancer. The works described above, while linking the loss of mitochondrial tumour suppressors to HIF activation, do not provide a biochemical rationale for the link.

The first biochemical explanation was reported in Selak et al. Succinate is a dicarboxylic acid, capable of crossing the mitochondrial inner membrane the only real barrier to small metabolites in the mitochondria via the dicarboxylate carrier.

Selak et al. Several studies have linked PHD activity to cancer formation owing to the loss of mitochondrial tumour suppressors. In this work, it was shown that, like succinate and potentially even better , fumarate, which accumulates in FH-deficient tumours, inhibits PHD activity in the cytosol.

Fumarate is not a product of PHD but it is chemically similar to succinate. Structurally, owing to a double bond in the centre of the dicarboxylic acid, fumarate is a rigid molecule compared to succinate. It is possible that due to its structure, fumarate interacts better with PHDs. Subsequent support for the biochemical role of succinate and fumarate in SDH- and FH-deficient tumours came from metabolomic studies of these tumours Pollard et al. Although SDHA was not identified as a tumour suppressor, bi-allelic mutations of SDHA were described in Leigh syndrome, an early-onset, progressive neurodegenerative disease cased by defective mitochondrial bioenergetics Rustin and Rotig, This study also addressed the important question as to why SDHA has never been reported to be a tumour suppressor gene and, in particular, why carriers of an SDHA mutant allele heterozygous parents or siblings of Leigh syndrome patients do not develop HPGL.

Briere et al. This indicates that for SDHA mutation carriers, the unlikely situation of inactivating three additional alleles, two of which are the intact wild-type alleles of one gene, needs to occur in order to develop HPGL.

Most tumours eventually reach a hypoxic state and it is conceivable that during rapid proliferation, tumour cells frequently experience intermittent hypoxic conditions.

Therefore, tumours that have adapted to an anaerobic state could have a survival advantage. Another aspect of adaptation to hypoxia is HIF-mediated neovascularization, seen in pseudohypoxic tumours such as those with SDH or FH deficiency see above.

This may be important for boosting the nutrient supply needed for tumour survival and growth. Lastly, as mentioned above, HIF plays a major role in the enhanced glycolysis observed in many tumours.

In addition to adaptation to anaerobic conditions, enhanced glycolysis is an energetic boost to the cells, rapidly generating ATP in the cytosol. Moreover, enhanced glucose breakdown provides building blocks for the synthesis of nucleotides via the pentose phosphate pathway , and amino and fatty acids from glycolytic and TCA cycle intermediates Figure 1 Owen et al. Even under conditions in which the TCA cycle is not fully functional, as is the case for SDH- and FH-deficient tumours, the anaplerotic reaction catalysed by pyruvate carboxylase, which converts pyruvate to oxaloacetate, can operate to replenish TCA cycle intermediates Figure 1.

Further metabolism of oxaloacetate by the TCA cycle generates precursors for many non-essential amino acids and for fatty acids and may provide reducing equivalents for oxidative phosphorylation. These processes are required for the anabolic reactions protein and DNA synthesis that lead to growth and proliferation of cancer cells. Whereas under normal conditions they provide the bulk of the energy needed to sustain cell life, mitochondria may also assume the role of executioner: when the elimination of cancer cells is required, either physiologically or therapeutically, mitochondria can trigger apoptosis, the programmed self-destruction of cells Green and Kroemer, Many pro- and anti-apoptotic proteins have been shown recently to affect mitochondrial physiology Hammerman et al.

For example, mitochondria-associated hexokinase, a key glycolytic enzyme, confers resistance to apoptosis Robey and Hay, Therefore, defective mitochondrial physiology, compensated for by enhanced glycolysis, could possibly render tumour cells apoptosis resistant. More direct evidence for the role of mitochondrial tumour suppressors in apoptosis has recently emerged from studies of VHL Lee et al.

The tumour pattern of VHL patients includes central nervous system and retinal hemangioblastomas, clear cell renal cell carcinoma and phaeochromocytoma. Lee et al. This indicates that, unlike the case of haemangioblastoma and clear cell renal cell carcinoma, mechanisms other than pseudo-hypoxia play a role in phaeochromocytoma development. It remains unclear, however, which PHD3 substrates cause the apoptotic response.

Apart from the metabolic signalling, which is mediated by succinate and fumarate, other mitochondria-derived molecules have been suggested to trigger the oncogenic signal. It seems likely that when tumorigenic mutations in the SDH subunits occur, succinate may still be oxidized to fumarate, but electrons accumulate on FAD and are then transferred to molecular oxygen to generate superoxide. Interestingly, in addition to mutagenesis, ROS may have another role in the pathology of these tumours.

Reactive oxygen species were reported to inhibit PHD activity under normoxic conditions by oxidizing the PHD cofactors ferrous iron and ascorbate Figure 3 Gerald et al.

This indicates that the SDH complex in these tumours is incapable of oxidizing succinate, a required enzymatic reaction for ROS generation. In the relatively short time since the discovery of mitochondrial tumour suppressors, vast genetic and biochemical knowledge has accumulated to help elucidate the pathways leading to tumorigenesis.

But, as is usually the case, we still have more questions than answers. The metabolic signalling pathway mediated by succinate or fumarate points to a common tumorigenic cause in SDH- or FH-deficient cells. This is in line with the physiological role of these enzymes in the TCA cycle. However, this metabolic signalling pathway does not explain the different tumour types that develop owing to mutations in either protein.

Despite their shared metabolic pathway, two major biochemical differences can help to tell SDH- and FH-mediated tumorigenesis apart. Although both molecules were shown to mediate HIF induction Isaacs et al.

In line with this idea, it was shown that fumarate is a better inhibitor of PHD2 than succinate Isaacs et al. It is possible however, that fumarate cannot match succinate in inhibiting PHD3-dependent apoptosis, as is the case in phaeochromocytoma Lee et al. As discussed above, transporting electrons is a risky business and therefore, despite the lack of a clear redox stress signature in SDHB- and SDHD-deficient tumours, it is still important to thoroughly explore the role of ROS in these tumours.

Moreover, by examining the different pathways described in Figure 2 , it appears that some tumour suppressor genes have a curtailing effect on HIF activity. This strongly emphasizes the role that HIF plays in oncogenesis. Moreover, it may suggest that PHDs themselves are tumour suppressors though to date, there is no evidence of that. This may be due to a degree of redundancy in the activity of different PHDs. Interestingly, recent studies have identified specific familial mutations in PHD2 and pVHL, each of which leads to erythrocytosis associated with HIF induction, but none of which is associated with cancer Ang et al.

Perhaps, cancer in these cases is avoided because HIF induction is relatively low this was not yet evaluated or because pseudo-hypoxia alone is insufficient to induce tumorigenesis. Most of the data available today indicate that succinate- or fumarate-mediated inhibition of PHDs is fundamental to this process, but PHD inhibition engages more than one downstream pathway.

This might complicate translating our knowledge into an effective treatment. Although HIF, a downstream target of some PHDs, is a very attractive target for treating cancer in general Hewitson and Schofield, and several inhibitors have already been tested in preclinical trials Kung et al. Therefore, the SDH, thanks to its unique redox properties, may be a key enzyme to control UQ pool redox poise under these conditions [ 13 ]. Disruption of complex II activity should alter TCA cycle metabolite levels in the mitochondrial matrix.

The succinate is the most efficient energy source, so the SDH activity assay can be an important method for measurement of the yeast vitality in scope to control, e. SDH activities can be measured in vitro in cell lysates or in mitochondrial fraction as well as in situ in individual cells. Since SDH is bound to the inner membrane, it is easily isolated along with the mitochondria by different techniques: sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method [ 20 ].

The mitochondrial fraction is the source of the enzyme. To use an artificial electron acceptor, the normal path of electrons through the mitochondrial electron transport system must be blocked.

This is accomplished by adding either sodium azide or potassium cyanide to the reaction mixture. These poisons inhibit the transfer of electrons from cytochrome a3 to the final electron acceptor, oxygen, thus electrons cannot be passed along by the preceding cytochromes and coenzyme Q. The reduction of DCIP can be followed spectrophotometrically since the oxidized form of the dye is blue and the reduced form is colorless.

This reaction can be summarized as. The change in absorbance, measured at nm, can be used to follow the reaction over time [ 21 ]. To use an artificial electron acceptor, the normal path of electrons in the electron transport chain must be blocked.

This is accomplished by adding either potassium cyanide or sodium azide to the reaction mixture. The rate of the disappearance of the blue color is proportional to the concentration of enzyme. The change in absorbance of the mixture is measured as a function of time and the enzyme concentration is determined from these data.

Enzymatic reactions in yeasts are usually studied in cell-free extracts which requires disruption of cells and as consequence, inactivation of particular enzymes often can be observed. Generally we can conclud that determination of SDH enzyme activity has proved to be a difficult enzyme to extract from respiratory membrane whilst still retaining its in vivo properties. Most of the described extraction procedures were rather drastic and yielded soluble preparations of rather dubious integrity [ 8 ].

In recent years quantitative histochemical procedures has been proved to be a powerful research tool, especially in microphotometric assessment in situ of the specific activity of dehydrogenases in individual cells. These assays are simple and valid alternative to conventional biochemical techniques. Methods in situ can provide the cellular resolution necessary to determine enzyme-specific activities not only in whole cell preparations but also in distinct subcellular compartments [ 19 ].

Reduction of various tetrazolium salts by dehydrogenases of metabolically active cells leads to production of highly colored end products — formazans Figure 7.

The history of the tetrazolium salts and formazans goes back years, to when Friese reacted benzene diazonium nitrate with nitromethane, to produce a cherry-red "Neue Verbindung". This was the first formazan. Nineteen years later, Von Pechmann and Runge oxidised a formazan to produce the first tetrazolium salt [ 21 ]. Tetrazolium salt and its coloured formazan. Many hundreds of tetrazolium salts and formazans were prepared in the following years, but only a handful have found applications in biological research.

There is a wide range of tetrazolium salts commonly used in the field of microbiology from the classical ones to the new generation of its derivatives. Among them are: blue tetrazolium chloride BT , 2,3,5-triphenyl tetrazolium chloride TTC , 3- 4,5-dimethylthiazolyl -2,5-diphenyltetrazolium bromide MTT , 5-cyano-2,3-ditolyl tetrazolium chloride CTC , 2,3-bis 2-methoxynitrosulphophenyl [ phenylamino carbonyl]-2H-tetrazolium hydroxide XTT , 4-[3- 4-idophenyl 4-nitrophenyl -2Htetrazolio]-1,3-benzene disulfonate WST1 , 2- p-iodophenyl -3 p-nitrophenyl phenyltetrazolium chloride INT or 2,2'-dibenzothiazolyl-5,5'-[4-di 2-sulfoethyl carbamoylphenyl]-3,3'- 3,3'-dimethoxy-4,4' biphenyl ditetrazolium, disodium salt WST-5 [ 19 , 22 , 23 ].

In the case of enzymatic reaction conducted in situ the plasma membrane forms a barrier with low degree of penetration. Therefore, cell permeabilization, e. According to the results obtained by Berlowska et al. After digitonin treatment, the visible formazan crystals were observed inside the yeast cells, but not outside them Figures 8 A, B. The formazan products are water-insoluble, but readily diffuses out of yeast cells after solubilization in DMSO.

Linear correlation was observed in the concentration range of yeast cells from 7 to 8 per sample. For yeast cell concentrations below 7 per sample the formazan color intensity signals were too low to detect with good precision. The results obtained for SDH activity were in good agreement. Yeast cells after reaction with blue tetrazolium chloride BT.

A — without permeabilization; B — with permeabilization by 0. Images of light microscopy. Yeast cells after reaction with 2,3,5-triphenyl tetrazolium chloride TTC. Images of fluorescence microscopy. Yeast cell after reaction with 2,3,5-triphenyl tetrazolium chloride TTC. Images of scanning microscopy. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of tested yeast strains [ 19 , 23 ]. Saccharomyces cerevisiae is a simple eukaryotic organism, with a complete genome sequence.

Many genetic tools that have been created during these years, including the complete collection of gene deletions and a considerable number of mechanisms and pathways existing in higher eukaryotes was first studied and described in yeast. The study of mitochondrial functions and dysfunction is of special interest in yeast because it is in this organism that mitochondrial genetics and recombination have been discovered and that nucleomitochondrial interactions have been studied in-depth.

There are also specific reasons for choosing S. This organism is petite-positive, which can successfully grow in the absence of oxygen. Therefore it can lose its mitochondrial genome provided it is supplied with a substrate for fermentation.

Consequently, all mutations of the mitochondrial genome can be studied without cell lethality. It is genetically easy to transfer mitochondria from one nuclear genetic background to another via karyogamy. Additionally, mitochondria can be transformed making in vitro mutation analysis possible. The richness and ease of yeast molecular genetics opens big opportunities, and even the major difference existing between human and yeast mitochondrial genomes, i.

To review mitochondrial diseases may be a very difficult task because the definition might include different kinds of metabolic disorders or degenerative syndromes [ 24 ]. Moreover, some important aspects have been extensively reviewed and the reader might refer to very good recent articles by DiMauro and Garone [ 25 ] for historical aspects, by Wallace et al. The previous review by Schwimmer et al. SDH in yeast and human are very similar. In the last ten years, deficiencies in TCA cycle enzymes have been shown to cause a wide spectrum of human diseases.

For instance, mutation in the gene encoding fumarase is a rare cause of encephalomyopathy and a far more common cause of leiomyomas of the skin and uterus and of renal cancer Table 1. The TCA path dysfunction may also result from concurrent impairments in several steps of the cycle. The ratios between TCA enzymes are consistent for each mammalian tissues presumably reflecting their metabolic demand.

Consequently, in addition to the determination of residual absolute activities, estimation of ratios between enzyme activities is an effective means of detecting partial but potentially harmful deficiencies. When used to assess respiratory chain activities, this approach enabled the identification of several gene mutations, even in patients with partial respiratory chain deficiencies. At present, TCA enzyme activities are measured using a series of independent.

Primary deficiencies in TCA cycle enzymes in humans [ 22 ]. The limited set of assays allowing both measurement of all TCA enzyme activities and detection of abnormalities in enzyme activity ratios were developed. These assays were used successfully to detect severe and partial isolated deficiencies in several TCA enzymes.

The reduction of DCPIP was measured using two wavelengths nm and nm with various substrates and the electron acceptors decylubiquinone and phenazine methosulfate.

The second assay measured -ketoglutarate dehydrogenase, aconitase, and isocitrate dehydrogenase activities. Hence, SDH 'inactivation' induces abnormal stimulation of the hypoxia-angiogenesis pathway. When complex II is absent, it can be disregarded as a source of additional superoxide production. Thus, the superoxide overproduction would lead to tumour formation that should be ascribed to the decreased ability of the SDH to adequately reduce the Q pool, a necessary condition to resist oxidative stress [ 8 ].

Ubiquinone, beside its function in the respiratory chain as an electron carrier mediating electron transfer between the various dehydrogenases and the cytochrome path, is working as a powerful antioxidant in biological membranes [ 13 ]. It is possibly for this exact reason in much larger amounts compared to other electron carriers of the respiratory chain, including the sum of the dehydrogenases.

When it is defective, the respiratory chain can produce an abnormal amount of superoxides involving additional respiratory chain components such as flavin radicals of complex I. Delivering electrons for the full reduction of Q to QH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria.

Therefore, the SDH is a key enzyme to control Q pool redox poise under these conditions, due to its unique redox properties [ 8 ]. Iron-sulfur Fe-S proteins facilitate multiple functions, including redox activity, enzymatic function, and maintenance of structural integrity. More than 20 proteins are involved in the biosynthesis of iron-sulfur clusters in eukaryotes.

Defective Fe-S cluster synthesis not only affects activities of many iron-sulfur enzymes, such as aconitase and succinate dehydrogenase, but also alters the regulation of cellular iron homeostasis, causing both mitochondrial iron overload and cytosolic iron deficiency.

Fe-S cluster biogenesis takes place essentially in every tissue of humans, and products of human disease genes have important roles in the process [ 40 ]. Succinate is an oxygen sensor in the cell and can help turn on specific pathways that stimulate cells to grow in a low-oxygen environment hypoxia.

In particular, succinate stabilizes a protein called hypoxia-inducible factor HIF by preventing a reaction that would allow HIF to be broken down. HIF controls several important genes involved in cell division and the formation of new blood vessels in a hypoxic environment.

However, a single mutation in the SDHA gene increases the risk that an individual will develop the condition, and additional mutation that deletes the normal copy of the gene is needed to cause tumor formation. This second mutation, called a somatic mutation, is acquired during a person's lifetime and is present only in tumor cells.

As a result, there is little or no SDH enzyme activity. Because the mutated SDH enzyme cannot convert succinate to fumarate, succinate accumulates in the cell.

The excess succinate abnormally stabilizes HIF, which also builds up in cells. Excess HIF stimulates cells to divide and triggers the production of blood vessels when they are not needed. Rapid and uncontrolled cell division, along with the formation of new blood vessels, can lead to the development of tumors.

Mutations in the SDHA gene were identified in a small number of people with Leigh syndrome, a progressive brain disorder that typically appears in infancy or early childhood. Affected children may experience vomiting, seizures, delayed development, muscle weakness, and problems with movement.

Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder. The one child died suddenly at the age of five months from a severe deterioration of neuromuscular, cardiac, and hepatic symptoms after an intermittent infection. These genetic changes disrupt the activity of the SDH enzyme, impairing the ability of mitochondria to produce energy. This suggested a role of additional nuclear genes involved in synthesis, assembly, or maintenance of SDH.

It is not known, however, how mutations in the SDHA gene are related to the specific features of Leigh syndrome [ 41 , 42 ]. Two plausible hypotheses have been proposed to explain the peculiar linkage between disruption of electron flow through mitochondrial complex II and tumorigenesis in neuroendocrine cells. Although certain mutations in these genes result in ROS production in Saccharomyces cerevisiae and mammalian cell lines, it is not clear that ROS accumulate to levels that are mutagenic.

The acetyl-CoA, has been oxidized to two molecules of carbon dioxide. Keep in mind that a reduction is really a gain of electrons. In the next SparkNote on Oxidative Phosphorylation and the electron transport chain, we will learn what processes take place to ultimately derive the the majority of the ATP we need to fuel our daily activity.

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