肿瘤SDH基因突变的研究进展

线粒体琥珀酸脱氢酶(succinate dehydrogenase,SDH)是三羧酸循环和有氧电子传递呼吸链中的关键酶之一,包含A、B、C、D 4个亚基.4个亚基分别由4个基因编码,即SDHA、SDHB、SDHC和SDHD,4个基因突变可以诱发癌症,包括副神经节瘤(paraganglioma,PGL)、嗜铬细胞瘤(pheochromocytoma,PHEO)、肾细胞癌(renal cell carcinoma,RCC)、胃肠道间质瘤(gastrointestinal stromal tumors,GIST)、Leigh综合症等.近年来,突变的SDH已经被证实是一种重要的诊断与预后的生物标志物和治疗分子标靶.本文就SDH存在的各种突变以及在肿瘤发生、发展与转移的作用机理研究的进展进行全面的论述.     

Activation of Brain Succinate Dehydrogenase by Lithium

IN spite of the widespread use of lithium salts as therapeutic and prophylactic agents against manic-depressive psychosis^{1, 2}, little is known about the mechanism of lithium effects^{3, 4}. Li⁺ stimulates noradrenergic neurones³, increases the uptake of this amine by isolated rat brain synaptosomes^{5, 6}, increases serotonin uptake into platelets obtained from manic-depressive patients⁷ and increases the levels of serotonin and tryptophan in rat brain⁸. On the other hand, Lallier observed inhibitory action of LiCl on dehydrogenase activity in homogenates of Rana fusca⁹. We have investigated whether long term treatment with Li⁺ affects brain succinate dehydrogenase activity.     

Calmodulin and the Regulation of Succinate Dehydrogenase Activity in the Mitochondria of Zea mays

Succinate dehydrogenase (SDH) was purified by DEAE C-32 chromatography from the mitochondrial fraction of corn ( Zea mays L. ). Free calmodulin (CAM) could not be detected in the purified SDH with the method based on the ability of SDH to stimulate NAD kinase, but it still contained some CAM when measured with the ELISA method. Purified SDH could stimulate NAD kinase only after heating to release free CAM. Plain polyacrylamide gel electrophoresis (PAGE) of the pufffled SDH revealed only one peptide band, but three peptide bands were shown on SDS-PAGE, their molecular weight being 67.0 kD, 30.0 kD, 16.7 kD respectively. The 67.0 kD and 30.0 kD peptides corresponded to the large and small molecular subunit of SDH respectively. The Rf value of the 16.7 kD peptide band was identical to the standard CAM in the SDS-PAGE. From all the above evidence, the authors suggested that CAM might exert its function of SDH regulation in a binding state with the SDH molecule.     

The Colorimetric Measurement of Succinate Dehydrogenase Activity in Subcellular Fractions

A 16K Sinclair ZXH1 equipped with an Analogue to Digital hoard and a Sinclair printer was used to capture data continuously from a total of eight temperature or light sensors. The paper describes the construction of the peripherals, explains how to connect them together, and provides a program to run the ZX81.     

Succinate Dehydrogenase is the Regulator of Respiration in Mycobacterium tuberculosis

In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.     

Characterization of the Excitotoxic Potential of the Reversible Succinate Dehydrogenase Inhibitor Malonate

Abstract: Although the mechanism of neuronal death in neurodegenerative diseases remains unknown, it has been hypothesized that relatively minor metabolic defects may predispose neurons to N -methyl- d -aspartate (NMDA) receptor-mediated excitotoxic damage in these disorders. To further investigate this possibility, we have characterized the excitotoxic potential of the reversible succinate dehydrogenase (SDH) inhibitor malonate. After its intrastriatal stereotaxic injection into male Sprague-Dawley rats, malonate produced a dose-dependent lesion when assessed 3 days after surgery using cytochrome oxidase histochemistry. This lesion was attenuated by coadministration of excess succinate, indicating that it was caused by specific inhibition of SDH. The lesion was also prevented by administration of the noncompetitive NMDA antagonist MK-801. MK-801 did not induce hypothermia, and hypothermia itself was not neuroprotective, suggesting that the neuroprotective effect of MK-801 was due to blockade of the NMDA receptor ion channel and not to any nonspecific effect. The competitive NMDA antagonist LY274614 and the glycine site antagonist 7-chlorokynurenate also profoundly attenuated malonate neurotoxicity, further indicating an NMDA receptor-mediated event. Finally, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline) was ineffective at preventing malonate toxicity at a dose that effectively reduced S -AMPA toxicity, indicating that non-NMDA receptors are involved minimally, if at all, in the production of the malonate lesion. We conclude that inhibition of SDH by malonate results in NMDA receptor-mediated excitotoxic neuronal death. If this mechanism of "secondary" or "weak" excitotoxicity plays a role in neurodegenerative disease, NMDA antagonists and other "antiexcitotoxic" strategies may have therapeutic potential for these diseases.     

Changes in Properties of Succinate Dehydrogenase during Senescence of Pea Cotyledons

Comparisons were made between succinate dehydrogenases (EC 1.3.99.1 [EC] )from 1-day-old and 5-day-old pea cotyledons. The enzyme wasloosely bound to the mitochondrial inner membrane in 5-day-oldcotyledons, but tightly in 1-day-old cotyledons. In addition,the enzyme partially purified from 5-day-old cotyledons wasmuch more labile than that from 1-day-old cotyledons. Succinaterapidly inactivated partially purified succinate dehydrogenasefrom 1-day-old cotyledons, but not from 5-day-old cotyledons.Dithiothreitol caused a change in the charge of the enzyme proteinfrom either 1- or 5-day-old cotyledons, only when succinatewas present. The enzyme from 5-day-old cotyledons differed fromthe succinate-induced labile form of the enzyme from 1-day-oldcotyledons in electrophoretic properties on a polyacrylamidegel. There was also a difference in the pattern of polyacrylamidegel electrophoresis between succinate dehydrogenases partiallypurified from 1- and 5-day-old cotyleodns. The partially purifiedenzyme from either 1- or 5-day-old cotyledons in the presenceof succinate had a molecular weight of 92,000. The molecularweight of the large subunit was suggested to be 65,000. Thepartially purified enzyme prepared from 1-day-old cotyledonsin the absence of succinate was in a form with a molecular weightof 113,000. (Received August 29, 1980; Accepted December 3, 1980)     

Genetic and Biochemical Characterization of Mutants of Bacillus subtilis Defective in Succinate Dehydrogenase

Eleven succinate-accumulating mutants of Bacillus subtilis have been mapped by transformation and transduction crosses and characterized with respect to activities of citric acid cycle enzymes. These mutants could be divided into three genetic groups. Nine of the mutants were found to map between argA and leu in the citF locus. A second group was located between lys-1 and trpC2 and the third group could not be located on the B. subtilis chromosome in extensive transduction crosses. All of the citF mutants lack detectable succinate dehydrogenase activity, whereas both of the other groups show a reduced level of this enzyme. In addition, most of the mutants in the citF locus lack cytochrome a, whereas the level of this cytochrome is normal in the other two groups. A procedure has been devised for the solubilization of the succinate dehydrogenase from the membrane of B. subtilis with the non-ionic detergent Brij 58. Some properties of the soluble and bound forms of succinate dehydrogenase are described.     

Mechanism of the Increase in Succinate Dehydrogenase Activity in Wounded Sweet Potato Root Tissue

The large subunit (mol wt: 65,000) of sweet potato succinatedehydrogenase was isolated by SDS-polyacrylamide gel electrophoresisof a succinate dehydrogenase preparation, which had been partiallypurified from root mitochondria by solubilizing the enzyme withEmulgen 810, DEAE-cellulose column chromatography, and polyacrylamidegel electrophoresis. Antibody to the purified large subunitwas produced in a rabbit, and the antiserum obtained was judgedto be specific to the large subunit based on the results ofdouble immunodiffusion tests and immunoelectrophoresis. Rocketimmunoelectrophoresis with the antiserum showed that the increasein succinate dehydrogenase activity during the ageing of sliced,sweet potato root tissue was due to an increase in the amountof enzyme protein. Both the increases in the activity of succinatedehydrogenase and in the amount of the large subunit proteinwere inhibited by cycloheximide or chloramphenicol. We proposethat synthesis of the large subunit of succinate dehydrogenaseon cytoplasmic ribosomes is controlled by a mitochondrial translationproduct(s). ¹ This work was supported in part by a research fund from TheIshida Foundation, Nagoya, Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Mutations in the C. elegans Succinate Dehydrogenase Iron-Sulfur Subunit Promote Superoxide Generation and Premature Aging

The mitochondrial succinate dehydrogenase (SDH) is an iron-sulfur flavoenzyme linking the Krebs cycle and the mitochondrial respiratory chain. Mutations in the human SDHB, SDHC and SDHD genes are responsible for the development of paraganglioma and pheochromocytoma, tumors of the head and neck or the adrenal medulla, respectively. In recent years, SDH has become recognized as a source of reactive oxygen species, which may contribute to tumorigenesis. We have developed a Caenorhabditis elegans model to investigate the molecular and catalytic effects of mutations in the sdhb-1 gene, which encodes the SDH iron-sulfur subunit. We created mutations in Pro211; this residue is located near the site of ubiquinone reduction and is conserved in human SDHB (Pro197), where it is associated with tumorigenesis. Mutant phenotypes ranged from relatively benign to lethal and were characterized by hypersensitivity to oxidative stress, a shortened life span, impaired respiration and overproduction of superoxide. Our data suggest that the SDH ubiquinone-binding site can become a source of superoxide and that the pathological consequences of SDH mutations can be mitigated with antioxidants, such as ascorbate and N-acetyl-l-cysteine. Our work leads to a better understanding of the relationship between genotype and phenotype in respiratory chain mutations and of the mechanisms of aging and tumorigenesis.     

Malonate Metabolism, Stimulation of Conidiation and Succinate Dehydrogenase Activity in Neurospora crassa

Malonate was studied for its effect on succinate dehydrogenase activity and conidiation. It Was found to stimulate the succinate dehydrogenase activity and also conidiation of Neurospora crassa. The efficiency of sucrose metabolization for cellular synthesis was improved in malonate supplemented cultures. High Concentration (0.5 M) had a distinct toxic effect on conidiation and economic efficiency. Teratological structures were observed at 0.5 M malonate concentration. A stimulation of the glyoxylate cycle is considered to be one of the factors responsible for the conidiogenic effect.     

Succinate Dehydrogenase Upregulation Destabilize Complex I and Limits the Lifespan of gas-1 Mutant

Many Caenorhabditis elegans mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (gas-1(fc21)) and long-lived (nuo-6(qm200)) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived gas-1(fc21) mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived gas-1(fc21) mutants and long-lived nuo-6(qm200) mutants also activate hypoxia-inducible factor –1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPR^mt). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, gas-1(fc21) mutant. We show that high levels of complex II negatively regulate longevity in gas-1(fc21) mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a “window” of mitochondrial dysfunction that allows lifespan prolongation.     

跖肌肌球蛋白腺苷三磷酸酶与琥珀酸脱氢酶染色的相关性探讨

研究大鼠、家兔和人跖肌的肌纤维型构成比例与分布。应用肌球蛋白腺苷三磷酸酶(ATP酶)和琥珀酸脱氢酶(SDH酶)染色法,观察比较各型肌纤维的组织化学特性。结果表明：肌球蛋白ATP酶染色,大鼠、家兔和人跖肌的肌纤维可分成I型、ⅡA型、ⅡB型肌纤维,横切面呈多边形或椭圆形,分别约占25％、35％和40％;琥珀酸脱氢酶染色,肌纤维呈蔚蓝色,以镶嵌交叉排列,可分为中染S0、深染FOG和浅染FG三型,分别约占25％、30％和45％,种系间无显著性差异。ATP酶活性反应肌纤维收缩的生理特性,而SDH活性反应肌纤维的代谢特征,两种分型的方法有所差异。     

The Presence of Two Forms of Succinate Dehydrogenase in Sweet Potato Root Mitochondria

Succinate dehydrogenase was partially purified from sweet potatoroot tissue by solubilization of the enzyme from the submitochondrialparticles, ammonium sulfate fractionation, and DEAE-cellulosecolumn chromatography. Sweet potato succinate dehydrogenaseexisted in two forms; these were separated by disc polyacrylamidegel electrophoresis or by hydroxyapatite column chromatography.There was a difference in the electric charge of the molecule,but not in the molecular weights of the two forms. No differencewas detected between the two forms of succinate dehydrogenasewith respect to their K_m values for succinate, pH-optimums andsubunit compositions. The two subunits that make up the enzymehave molecular weights of about 26,000 and 65,000. ¹ This work was supported in part by Grant-in-Aid 411308 forScientific Research from the Ministry of Education, Scienceand Culture of Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Flavinylation and Assembly of Succinate Dehydrogenase Are Dependent on the C-terminal Tail of the Flavoprotein Subunit

The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ∼70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg⁵⁸² in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg⁶³⁸ compromises SDH function only when present in combination with a Cys⁶³⁰ substitution. Mutations of either Arg⁵⁸² or Arg⁶³⁸/Cys⁶³⁰ do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.     

Activity and Catalytic Characteristics of Rat Liver Mitochondrial Succinate Dehydrogenase under Moderate Hypothermia

Biophysics - We have previously shown that short-term moderate (30°C) hypothermia contributed to a significant increase in the intensity of free radical processes and changes in a number of...     

Exacerbation of NMDA, AMPA, and l-Glutamate Excitotoxicity by the Succinate Dehydrogenase Inhibitor Malonate

Abstract: We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-d -aspartate (NMDA), S-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and l -glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm³), as did coinjection of malonate with S-AMPA (11.0 ± 1.0 mm³) or glutamate (12.8 ± 0.7 mm³). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm³). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 ± 0.7 mm³), but it attenuated the toxicity of malonate plus glutamate by ～40% (7.5 ± 0.9 mm³). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm³, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 ± 0.9 mm³). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Succinate Dehydrogenase, Monoamine Oxidase and Glutamine Synthetase in Developing Human Foetal Brain Regions

In human foetal brain ontogeny the cerebral activity of succinate oxidoreductase (EC 1.3.99.1), i.e. succinate dehydrogenase (SDH), is higher than the cerebellar activity. With rise in foetal body weight the activity in all the brain regions gradually declines. SDH in all the brain regions shows two high-activity periods, one at 20-35 g and another at 110-220 g body weight. The enzyme exhibits a craniocaudal pattern of development. At all times of gestation, L-glutamate:ammonia ligase (EC 6.3.1.2), i.e. glutamine synthetase, activity in the spinal cord and medulla is higher than in the other three regions. At 190 g body weight glutamine synthetase shows an activity peak in all brain regions. Monoamine:oxygen oxidoreductase (EC 1.4.3.4). i.e. monoamine oxidase (MAO), is present much before the onset of electrical activity. It develops caudocranially and exhibits a biphasic pattern of development in all the regions. It increases considerably in the medulla and the spinal core towards late gestational periods.     

甜菜碱醛脱氢酶在菠菜叶细胞中的定位

将菠菜叶片匀浆后．用差速离心和梯度率心分离叶绿体、过氧物酶体、微粒体等细胞器和１０００００×ｇ上清法部分。用酶活测定法测定各部分甜菜碱醛脱氢酶（ＢＡＤＨ）的活性;用免疫扩散法鉴定各组分的ＢＡＤＨ。除叶绿体外,过氧物酶体、微粒体．以及１０００００×ｇ上清液中也存在ＢＡＤＨ。