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PubMed2002

Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck paragangliomas.

BACKGROUND: Paragangliomas are rare and highly heritable tumours of neuroectodermal origin that often develop in the head and neck region. Germline mutations in the mitochondrial complex II genes, SDHB, SDHC, and SDHD, cause hereditary paraganglioma (PGL). METHODS: We assessed the frequency of SDHB, SDHC, and SDHD gene mutations by PCR amplification and sequencing in a set of head and neck paraganglioma patients who were previously managed in two otolaryngology clinics in the USA. RESULTS: Fifty-five subjects were grouped into 10 families and 37 non-familial cases. Five of the non-familial cases had multiple tumours. Germline SDHD mutations were identified in five of 10 (50%) familial and two of 37 ( approximately 5%) non-familial cases. R38X, P81L, H102L, Q109X, and L128fsX134 mutations were identified in the familial cases and P81L was identified in the non-familial cases. Both non-familial cases had multiple tumours. P81L and R38X mutations have previously been reported in other PGL families and P81L was suggested as a founder mutation. Allelic analyses of different chromosomes carrying these mutations did not show common disease haplotypes, strongly suggesting that R38X and P81L are potentially recurrent mutations. Germline SDHB mutations were identified in two of 10 (20%) familial and one of 33 ( approximately 3%) non-familial cases. P131R and M71fsX80 were identified in the familial cases and Q59X was identified in the one non-familial case. The non-familial case had a solitary tumour. No mutations could be identified in the SDHC gene in the remaining four families and 20 sporadic cases. CONCLUSIONS: Mutations in SDHD are the leading cause of head and neck paragangliomas in this clinic patient series. SDHD and SDHB mutations account for 70% of familial cases and approximately 8% of non-familial cases. These results also suggest that the commonness of the SDHD P81L mutation in North America is the result of both a founder effect and recurrent mutations.

Alleles↗

PubMed1996

Involvement of the mitochondrion respiratory chain in the synergy achieved by treatment of human ovarian carcinoma cell lines with both tumor necrosis factor-alpha and cis-diamminedichloroplatinum.

BACKGROUND: Previous studies have demonstrated that treatment of human tumor cell lines with a combination of cis-diamminedichloroplatinum (CDDP) and tumor necrosis factor-alpha (TNF-alpha) results in additive/synergistic cytotoxic effects and reverses tumor cell resistance to TNF drugs. Free radical intermediates are induced by both TNF-alpha and CDDP; however, the role of free radicals in synergy is not known. This study investigated the effect of two inhibitors on synergy, phenoxan (Phe) and butylated hydroxyanisole (BHA), which inhibit Complex I and Complex I and II of the mitochondrion respiratory chain, respectively. METHODS: Three human ovarian carcinoma cell lines of different sensitivity to TNF-alpha and/or CDDP were selected for the study and consisted of 222, a TNF/CDDP-sensitive line, 222TR (TNF-resistant), a TNF-resistant, CDDP-sensitive line, and AD10, a TNF-sensitive, CDDP-resistant line. Cytotoxicity was determined by the microculture tetrazolium dye assay. RESULTS: Synergy in cytotoxicity was achieved in all three lines treated with a combination of TNF-alpha and CDDP. Cytotoxicity by either TNF-alpha or CDDP or by both TNF-alpha and CDDP was inhibited in the presence of either Phe or BHA. Pretreatment of tumor cells with either Phe or BHA for up to 4 hours, washed and followed by the addition of the cytotoxic agents (alone or combined), resulted in no inhibitory effect. Pretreatment of the cells with the cytotoxic agent for up to 2 hours, washed and then followed by the addition of Phe, resulted in significant inhibition of cytotoxicity. In contrast to Phe, the addition of BHA as late as 12 hours post pretreatment of the cells with the cytotoxic agent(s) still inhibited cytotoxicity. These results demonstrated that free radicals are involved in cytotoxicity mediated by a single agent, and in synergy with both agents. Further, the results demonstrated that Phe acts at an early stage of the cytotoxic pathway and that BHA acts at both an early and a late stage of the cytotoxic pathway. CONCLUSIONS: These results demonstrated that both TNF-alpha and CDDP rapidly stimulate the induction of free radicals but a lag of several hours was necessary to initiate the irreversible program of cell death. Further, the studies demonstrated that synergy and reversal of drug resistance in ovarian tumor cells by TNF-alpha and CDDP, used in combination, share the same pathway of cytotoxicity as that mediated by TNF-alpha or CDDP used as a single agent.

Antineoplastic Agents↗

PubMed1997

ADP-regulation of mitochondrial free radical production is different with complex I- or complex II-linked substrates: implications for the exercise paradox and brain hypermetabolism.

In agreement with classic studies, succinate-supplemented rat and pigeon heart and nonsynaptic brain mitochondrial free radical production is stopped by ADP additions causing the stimulation of respiration from State 4 to State 3. Nevertheless, with Complex I-linked substrates, mitochondria produce free radicals in State 3 at rates similar or somewhat higher than during resting respiration. The absence of sharp increases in free radical production during intense respiration is possible due to strong decreases of free radical leak in State 3. The results indicate that Complex I is the main mitochondrial free radical generator in State 3, adding to its already known important generation of active oxygen species in State 4. The observed rate of mitochondrial free radical production with Complex I-linked substrates in the active State 3 can help to explain two paradoxes: (a) the lack of massive muscle oxidative damage and shortening of life span due to exercise, in spite of up to 23-fold increases of oxygen consumption together with the very low levels of antioxidants present in heart, skeletal muscle, and brain; (b) the presence of some degree of oxidative stress during exercise and hyperactivity in spite of the stop of mitochondrial free radical production by ADP with succinate as substrate.

Adenosine Diphosphate↗

PubMed1999

Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli.

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.

Anilides↗

PubMed1998

The Saccharomyces cerevisiae TCM62 gene encodes a chaperone necessary for the assembly of the mitochondrial succinate dehydrogenase (complex II).

The assembly of the mitochondrial respiratory chain is mediated by a large number of helper proteins. To better understand the biogenesis of the yeast succinate dehydrogenase (SDH), we searched for assembly-defective mutants. SDH is encoded by the SDH1, SDH2, SDH3, and SDH4 genes. The holoenzyme is composed of two domains. The membrane extrinsic domain, consisting of Sdh1p and Sdh2p, contains a covalent FAD cofactor and three iron-sulfur clusters. The membrane intrinsic domain, consisting of Sdh3p and Sdh4p, is proposed to bind two molecules of ubiquinone and one heme. We isolated one mutant that is respiration-deficient with a specific loss of SDH oxidase activity. SDH is not assembled in this mutant. The complementing gene, TCM62 (also known as SCYBR044C), does not encode an SDH subunit and is not essential for cell viability. It encodes a mitochondrial membrane protein of 64,211 Da. The Tcm62p sequence is 17.3% identical to yeast hsp60, a molecular chaperone. The Tcm62p amino terminus is in the mitochondrial matrix, whereas the carboxyl terminus is accessible from the intermembrane space. Tcm62p forms a complex containing at least three SDH subunits. We propose that Tcm62p functions as a chaperone in the assembly of yeast SDH.

Amino Acid Sequence↗

PubMed2001

Abortive assembly of succinate-ubiquinone reductase (complex II) in a ferrochelatase-deficient mutant of Escherichia coli.

Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.

Cell Membrane↗

PubMed2003

1,3-Dinitrobenzene inhibits mitochondrial complex II in rat and mouse brainstem and cortical astrocytes.

1,3-Dinitrobenzene (DNB) produces edematous, glio-vascular lesions that are initially confined to brainstem nuclei with high energy requirements in rats and mice. Perturbation of energy producing processes in the cell is known to induce formation of the mitochondrial permeability transition pore (mtPTP) complex. Selective vulnerability of brainstem astrocytes to DNB is mediated by a 10-fold lower threshold for opening of the cyclosporin A-inhibitable mitochondrial permeability transition (MPT) pore than their cortical counterparts. Other nitrocompounds, such as 3-nitropropionic acid, selectively interfere with regional energy metabolism, including mitochondrial succinate dehydrogenase activity. However, the link between DNB-induced onset of the MPT and disruption of energy producing processes in the astrocyte remains unclear. The effects of DNB on succinate dehydrogenase activity were evaluated in cultured neonatal rat and mouse brainstem and cortical astrocytes. Both histochemical and spectrophotometric assays confirmed significant temporal inhibition of SDH activity in brainstem and cortical astrocytes 0.5, 2 and 5h following exposure to 100 microM DNB in vitro. Although DNB-induced inhibition of SDH was significantly decreased by CsA pretreatment in brainstem astrocytes after 0.5 and 2h and with a second pore inhibitor, bongkrekic acid (BKA) after 5h, both inhibitors failed to reduce inhibition of SDH activity in cortical astrocytes. These data suggest that DNB-induced inhibition of SDH may be independent of differential regional activation of the mtPTP complex in astrocytes and that an unidentified cyclosporin A-inhibitable factor mediates DNB-induced loss of SDH function.

Animals↗

PubMed1980

Studies on the succinate dehydrogenating system. II. Reconstitution of succinate-ubiquinone reductase from the soluble components.

1. A protein fraction containing three polypeptides (the major one with Mr < 13 000) was isolated by means of Triton X-100 extraction of submitochondrial particles specifically treated to remove succinate dehydrogenase. 2. The mixing of the protein fraction with the soluble reconstitutively active succinate dehydrogenase results in formation of highly active succinate-DCIP reductase which is sensitive to thenoyltrifluoroacetone or carboxin. 3. The maximal turnover number of succinate dehydrogenase in the succinate-DCIP reductase reaction revealed in the presence of a saturating amount of the protein fraction is slightly higher than that measured with phenazine methosulfate as artificial electron acceptor. 4. The protein fraction greatly increases the stability of soluble succinate dehydrogenase under aerobic conditions. 5. The titration of soluble succinate dehydrogenase by the protein fraction shows that smaller amounts of the protein fraction are required to block the reduction of ferrycyanide by Hipip center than that required to reveal the maximal catalytic capacity of the enzyme. 6. The apparent Km of the reconstituted system for DCIP depends on the amount of protein fraction; the more protein fraction added to the enzyme, the lower the Km value obtained. 7. A comparison of different reconstituted succinate-ubiquinone reductases described in the literature is presented and the possible arrangement of the native and reconstituted succinate-ubiquinone region of the respiratory chain is discussed.

2,6-Dichloroindophenol↗

PubMed2004

Chromosome 11 monosomy in conjunction with a mutated SDHD initiation codon in nonfamilial paraganglioma cases.

Paragangliomas of the head and neck region are a group of rare, usually benign, slow-growing tumors developing from paraganglionic chemoreceptors in most patients. Mutations in a subunit of the mitochondrial enzyme II complex (succinate dehydrogenase [SDHD]) were shown to be responsible for the formation of paragangliomas. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), was observed recently. We analyzed DNA derived from tumor sections of three unrelated paraganglioma patients (one case with multiple paragangliomas, two cases with single tumors; all of them sporadic cases) for mutations in the SDHD gene by direct sequencing. Microsatellite-based LOH was performed, and events of chromosomal loss were validated by fluorescence in situ hybridization (FISH) on paraffin-embedded tumor and normal tissue by using centromeric satellite DNA. Sequence analysis revealed mutations in SDHD exon 1 in all patients, affecting the initiation codon (M1V). Another alteration was detected in exon 2 but was lacking in tumor DNA and therefore classified as polymorphism (H50R). LOH and FISH analyses demonstrated partial/total monosomy for chromosome 11 in the tumor samples tested. A common genetic mechanism appears to be the pathophysiologic basis for sporadic tumor development because the proposed two-hit model comprising both LOH and point mutation is manifest in our patients. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors in one of the patients.

Adult↗

PubMed1998

Molecular genetics of succinate:quinone oxidoreductase in eukaryotes.

Succinate:quinone oxidoreductase is a membrane-associated complex in mitochondria, often referred to as complex II, based on the fractionation scheme developed by Y. Hatefi and colleagues. It consists of four peptides, two of which are integral membrane proteins (15 and 12-13 kDa, respectively) and two others that are peripheral membrane proteins, i.e., a flavoprotein (Fp, 70 kDa) and an iron-protein (Ip, 27 kDa). The mature, functional complex contains a cytochrome in association with the membrane proteins, a flavin linked covalently to the largest peptide, and three iron-sulfur clusters in the 27-kDa subunit. The present review touches only briefly on the biochemical and biophysical properties of this complex. Instead, the focus is on the molecular-genetic studies that have become possible since the first genes from eukaryotes were cloned in 1989. The evolutionary conservation of the amino acid sequence of both the Fp and the Ip peptides has facilitated the cloning of these genes from a large variety of eukaryotic organisms by PCR-based methods. The review addresses questions related to the regulation of the expression of these genes, with an emphasis on mammals and yeast, for which most of the information is available. Four different genes have to be co-ordinately regulated. Transcriptional as well as posttranscriptional regulatory mechanisms have been observed in diverse organisms. Intriguing observations have been made in studies of this enzyme during the life cycle of organisms existing alternately under aerobic and anaerobic conditions. Naturally occurring or induced mutations in these genes have shed light on several questions related to the assembly of this complex, and on the relationship between structure and function. Four different peptides are imported into the mitochondria. They have to be modified, folded, and assembled. The stage is set for the exploration of highly specific changes introduced by site-directed mutagenesis. Until recently the genes were believed to be exclusively nuclear in all eukaryotes, but exceptions have since been found. This finding has relevance in the discussion of the evolution of mitochondria from prokaryotes. A highly conserved set of genes is found in prokaryotes, and some informative comparisons on gene organization and expression in prokaryotes and eukaryotes have been included.

Amino Acid Sequence↗

PubMed1998

Methylmalonate toxicity in primary neuronal cultures.

Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.

Adenosine Diphosphate↗

PubMed2001

Retention of heme in axial ligand mutants of succinate-ubiquinone xxidoreductase (complex II) from Escherichia coli.

Succinate-ubiquinone oxidoreductase (SdhCDAB, complex II) from Escherichia coli is a four-subunit membrane-bound respiratory complex that catalyzes ubiquinone reduction by succinate. In the E. coli enzyme, heme b(556) is ligated between SdhC His(84) and SdhD His(71). Contrary to a previous report (Vibat, C. R. T., Cecchini, G., Nakamura, K., Kita, K., and Gennis, R. B. (1998) Biochemistry 37, 4148-4159), we demonstrate the presence of heme in both SdhC H84L and SdhD H71Q mutants of SdhCDAB. EPR spectroscopy reveals the presence of low spin heme in the SdhC H84L (g(z) = 2.92) mutant and high spin heme in the SdhD H71Q mutant (g = 6.0). The presence of low spin heme in the SdhC H84L mutant suggests that the heme b(556) is able to pick up another ligand from the protein. CO binds to the reduced form of the mutants, indicating that it is able to displace one of the ligands to the low spin heme of the SdhC H84L mutant. The g = 2.92 signal of the SdhC H84L mutant titrates with a redox potential at pH 7.0 (E(m)(,7)) of approximately +15 mV, whereas the g = 6.0 signal of the SdhD H71Q mutant titrates with an E(m)(,7) of approximately -100 mV. The quinone site inhibitor pentachlorophenol perturbs the heme optical spectrum of the wild-type and SdhD H71Q mutant enzymes but not the SdhC H84L mutant. This finding suggests that the latter residue also plays an important role in defining the quinone binding site of the enzyme. The SdhC H84L mutation also results in a significant increase in the K(m) and a decrease in the k(cat) for ubiquinone-1, whereas the SdhD H71Q mutant has little effect on these parameters. Overall, these data indicate that SdhC His(84) has an important role in defining the interaction of SdhCDAB with both quinones and heme b(556).

Benzoquinones↗

PubMed1957