The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase*

PubMed Central

Glasser, Nathaniel R.; Wang, Benjamin X.; Hoy, Julie A.; Newman, Dianne K.

2017-01-01

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa. Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG. Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism. PMID:28174304

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

PubMed

Glasser, Nathaniel R; Wang, Benjamin X; Hoy, Julie A; Newman, Dianne K

2017-03-31

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro , suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD + (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD + in LpdG and other enzymes, achieving the same end by a different mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

PubMed Central

Sazanov, Leonid A.; Burrows, Paul A.; Nixon, Peter J.

1998-01-01

The plastid genomes of several plants contain ndh genes—homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways. PMID:9448329

Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome.

PubMed

Bénit, P; Slama, A; Cartault, F; Giurgea, I; Chretien, D; Lebon, S; Marsac, C; Munnich, A; Rötig, A; Rustin, P

2004-01-01

Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.

Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy.

PubMed

Vacchina, Paola; Lambruschi, Daniel A; Uttaro, Antonio D

2018-03-01

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC 50 values one order of magnitude lower (62-66 μM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [ 14 C]-octanoic acid was taken up 12 fold less efficiently than [ 14 C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments. Copyright © 2018 Elsevier Inc. All rights reserved.

Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting.

PubMed

Dung, Vuu My; Suong, Dang Ngoc Anh; Okamaoto, Yuji; Hiramatsu, Yu; Thao, Dang Thi Phuong; Yoshida, Hideki; Takashima, Hiroshi; Yamaguchi, Masamitsu

2018-05-15

Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit beta (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. In this study, we established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which were consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy. Copyright © 2018 Elsevier Inc. All rights reserved.

Pyruvate dehydrogenase complex (PDC) export from the mitochondrial matrix.

PubMed

Ng, Fanny; Tang, Bor Luen

2014-01-01

Studies on mitochondria protein import had revealed in detail molecular mechanisms of how peptides and proteins could be selectively targeted and translocated across membrane bound organelles. The opposite process of mitochondrial export, while known to occur in various aspects of cellular physiology and pathology, is less well understood. Two very recent reports have indicated that a large mitochondrial matrix protein complex, the pyruvate dehydrogenase complex (PDC) (or its component subunits), could be exported to the lysosomes and the nucleus, respectively. In the case of the latter, evidence was presented to suggest that the entire complex of 8-10 MDa could translocate in its entirety from the mitochondrial matrix to the nucleus upon mitogenic or stress stimuli. We discuss these findings in perspective to what is currently known about the processes of transport in and out of the mitochondrion.

Structure and function of complex I in animals and plants - a comparative view.

PubMed

Senkler, Jennifer; Senkler, Michael; Braun, Hans-Peter

2017-09-01

The mitochondrial NADH dehydrogenase complex (complex I) has a molecular mass of about 1000 kDa and includes 40-50 subunits in animals, fungi and plants. It is composed of a membrane arm and a peripheral arm and has a conserved L-like shape in all species investigated. However, in plants and possibly some protists it has a second peripheral domain which is attached to the membrane arm on its matrix exposed side at a central position. The extra domain includes proteins resembling prokaryotic gamma-type carbonic anhydrases. We here present a detailed comparison of complex I from mammals and flowering plants. Forty homologous subunits are present in complex I of both groups of species. In addition, five subunits are present in mammalian complex I, which are absent in plants, and eight to nine subunits are present in plant complex I which do not occur in mammals. Based on the atomic structure of mammalian complex I and biochemical insights into complex I architecture from plants we mapped the species-specific subunits. Interestingly, four of the five animal-specific and five of the eight to nine plant-specific subunits are localized at the inner surface of the membrane arm of complex I in close proximity. We propose that the inner surface of the membrane arm represents a workbench for attaching proteins to complex I, which are not directly related to respiratory electron transport, like nucleoside kinases, acyl-carrier proteins or carbonic anhydrases. We speculate that further enzyme activities might be bound to this micro-location in other groups of organisms. © 2017 Scandinavian Plant Physiology Society.

Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

NASA Astrophysics Data System (ADS)

Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Ogorzalek Loo, Rachel R.; Loo, Joseph A.

2014-12-01

"Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

Clostridium acidurici electron-bifurcating formate dehydrogenase.

PubMed

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Thauer, Rudolf K

2013-10-01

Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD(+) and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2.

Decreased enzyme activity and contents of hepatic branched-chain alpha-keto acid dehydrogenase complex subunits in a rat model for type 2 diabetes mellitus.

PubMed

Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Sato, Yuzo; Shimomura, Yoshiharu

2009-10-01

The mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) is responsible for the committed step in branched-chain amino acid catabolism. In the present study, we examined BCKDC regulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats both before (8 weeks of age) and after (25 weeks of age) the onset of type 2 diabetes mellitus. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. Plasma branched-chain amino acid and branched-chain alpha-keto acid concentrations were significantly increased in young and middle-aged OLETF rats. Although the hepatic complex was nearly 100% active in all animals, total BCKDC activity and protein abundance of E1alpha, E1beta, and E2 subunits were markedly lower in OLETF than in LETO rats at 8 and 25 weeks of age. In addition, hepatic BCKDC activity and protein amounts were significantly decreased in LETO rats aged 25 weeks than in LETO rats aged 8 weeks. In skeletal muscle, E1beta and E2 proteins were significantly reduced, whereas E1alpha tended to increase in OLETF rats. Taken together, these results suggest that (1) whole-body branched-chain alpha-keto acid oxidation capacity is extremely reduced in OLETF rats independently of diabetes development, (2) the aging process decreases BCKDC activity and protein abundance in the liver of normal rats, and (3) differential posttranscriptional regulation for the subunits of BCKDC may exist in skeletal muscle.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Colombo, I.; Volta, M.

The electron-transfer flavoprotein (ETF), located in the mitochondrial matrix, is a nuclear-encoded enzyme delivering to the respiratory chain electrons by straight-chain acyl-CoA dehydrogenases and other dehydrogenases. ETF is composed of a 35-kDa [alpha]-subunit that is cleaved to a 32-kDa protein during mitochondrial import (ETFA) and a [beta]-subunit that reaches the mitochondrion unmodified (ETFB). The cDNA encoding both these subunits has been cloned and sequenced. 14 refs., 1 fig.

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

PubMed Central

Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

1991-01-01

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

PubMed Central

Sanz, Alberto; Soikkeli, Mikko; Portero-Otín, Manuel; Wilson, Angela; Kemppainen, Esko; McIlroy, George; Ellilä, Simo; Kemppainen, Kia K.; Tuomela, Tea; Lakanmaa, Matti; Kiviranta, Essi; Stefanatos, Rhoda; Dufour, Eric; Hutz, Bettina; Naudí, Alba; Jové, Mariona; Zeb, Akbar; Vartiainen, Suvi; Matsuno-Yagi, Akemi; Yagi, Takao; Rustin, Pierre; Pamplona, Reinald; Jacobs, Howard T.

2010-01-01

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling. PMID:20435911

The crystallogenesis of a human estradiol dehydrogenase-substrate complex

NASA Astrophysics Data System (ADS)

Zhu, Dao-Wei; Azzi, Arezki; Rehse, Peter; Lin, Sheng-Xiang

1996-10-01

Human 17β-hydroxysteroid dehydrogenase type 1 is an important steroidogenic enzyme catalyzing the synthesis of the most active estrogen: estradiol. The enzyme is formed by two identical subunits (34.5 kDa). In this paper, we report the preparation of a stoichiometric 17β-HSD1-estradiol complex sample at a much higher concentration than the solubility of the free substrate, using a gradual concentration of the enzyme-substrate mixture starting at low concentration. The complex is successfully crystallized by vapor diffusion at pH 7.5 with polyethyleneglycol 4000 as the precipitating agent. The space group is C2 with a = 123.56 Å, b = 45.21 Å, c = 61.30 Å and β = 99.06°. There is one monomer in the asymmetric unit and two molecules of the enzyme in a unit cell. A diffraction data set to 2.5 Å has been collected to 86% completeness on native crystals. The high quality of the electronic density map of estradiol supports the full occupancy of the binding site, thus confirming the efficiency of the complex preparation. This method will also be useful in crystallizing other steroid-dehydrogenase complexes.

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli.

PubMed

Bagramyan, K; Trchounian, A

2003-11-01

Formate hydrogen lyase from Escherichia coli is a membrane-bound complex that oxidizes formic acid to carbon dioxide and molecular hydrogen. Under anaerobic growth conditions and fermentation of sugars (glucose), it exists in two forms. One form is constituted by formate dehydrogenase H and hydrogenase 3, and the other one is the same formate dehydrogenase and hydrogenase 4; the presence of small protein subunits, carriers of electrons, is also probable. Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel-cobalt). Its formation also depends on the external pH and the presence of formate. Activity of both forms requires F(0)F(1)-ATPase; this explains dependence of the complex functioning on proton-motive force. It is also possible that the formate hydrogen lyase complex will exhibit its own proton-translocating function.

Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

PubMed Central

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

2013-01-01

Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD+ and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2. PMID:23872566

Crystal structure studies of NADP{sup +} dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, S.M.; Pampa, K.J.; Manjula, M.

2014-06-20

Highlights: • We determined the structure of isocitrate dehydrogenase with citrate and cofactor. • The structure reveals a unique novel terminal domain involved in dimerization. • Clasp domain shows significant difference, and catalytic residues are conserved. • Oligomerization of the enzyme is quantized with subunit-subunit interactions. • Novel domain of this enzyme is classified as subfamily of the type IV. - Abstract: NADP{sup +} dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP{supmore » +} was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.« less

The molecular origins of specificity in the assembly of a multienzyme complex.

PubMed

Frank, René A W; Pratap, J Venkatesh; Pei, Xue Y; Perham, Richard N; Luisi, Ben F

2005-08-01

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.

Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site.

PubMed

Armstrong, Craig T; Anderson, J L Ross; Denton, Richard M

2014-04-15

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca2+, adenine nucleotides and NADH, with NADH exerting a major influence on the K0.5 value for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (D114ADLD) and site 2 (E139SDLD). Comparison of sequences from vertebrates with those from Ca2+-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca2+, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A114AALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with A139SALA) shows no measurable change in Ca2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca2+-binding site, which is distinct from other previously described Ca2+-binding sites.

Human Pyruvate Dehydrogenase Complex E2 and E3BP Core Subunits: New Models and Insights from Molecular Dynamics Simulations.

PubMed

Hezaveh, Samira; Zeng, An-Ping; Jandt, Uwe

2016-05-19

Targeted manipulation and exploitation of beneficial properties of multienzyme complexes, especially for the design of novel and efficiently structured enzymatic reaction cascades, require a solid model understanding of mechanistic principles governing the structure and functionality of the complexes. This type of system-level and quantitative knowledge has been very scarce thus far. We utilize the human pyruvate dehydrogenase complex (hPDC) as a versatile template to conduct corresponding studies. Here we present new homology models of the core subunits of the hPDC, namely E2 and E3BP, as the first time effort to elucidate the assembly of hPDC core based on molecular dynamic simulation. New models of E2 and E3BP were generated and validated at atomistic level for different properties of the proteins. The results of the wild type dimer simulations showed a strong hydrophobic interaction between the C-terminal and the hydrophobic pocket which is the main driving force in the intertrimer binding and the core self-assembly. On the contrary, the C-terminal truncated versions exhibited a drastic loss of hydrophobic interaction leading to a dimeric separation. This study represents a significant step toward a model-based understanding of structure and function of large multienzyme systems like PDC for developing highly efficient biocatalyst or bioreaction cascades.

Regulation of yeast central metabolism by enzyme phosphorylation

PubMed Central

Oliveira, Ana Paula; Ludwig, Christina; Picotti, Paola; Kogadeeva, Maria; Aebersold, Ruedi; Sauer, Uwe

2012-01-01

As a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes. PMID:23149688

Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

ERIC Educational Resources Information Center

Nash, Barbara T.

2007-01-01

SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex*

PubMed Central

Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S.; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

2014-01-01

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

PubMed

Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

2014-10-24

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue.

PubMed

Richard, Allison J; Hang, Hardy; Stephens, Jacqueline M

2017-12-01

STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene c ytokine- i nducible SH 2-containing protein ( cish ). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

PubMed Central

Clarkson, G H; Neagle, J; Lindsay, J G

1991-01-01

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1996968

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

PubMed

Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

2014-01-01

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

PubMed

Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

2013-11-01

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

Pyruvate dehydrogenase complexes from the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum, helminths exhibiting anaerobic mitochondrial metabolism.

PubMed

Diaz, F; Komuniecki, R W

1994-10-01

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from 2 parasitic helminths exhibiting anaerobic mitochondrial metabolism, the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum. The P. equorum PDC yielded 7 major bands when separated by SDS-PAGE. The bands of 72, 55-53.5, 41 and 36 kDa corresponded to E2, E3, E1 alpha and E1 beta, respectively. The complex also contained additional unidentified proteins of 43 and 45 kDa. Incubation of the complex with [2-14C]pyruvate resulted in the acetylation of only E2. These results suggest that the P. equorum PDC lacks protein X and exhibits an altered subunit composition, as has been described previously for the PDC of the related nematode, Ascaris suum. In contrast, the D. caninum PDC yielded only four major bands after SDS-PAGE of 59, 58, 39 and 34 kDa, which corresponded to E3, E2, E1 alpha and E1 beta, respectively. Incubation of the D. caninum complex with [2-14C]pyruvate resulted in the acetylation of E2 and a second protein which comigrated with E3, suggesting that the D. caninum complex contained protein X and had a subunit composition similar to PDCs from other eukaryotic organisms. Both helminth complexes appeared less sensitive to inhibition by elevated NADH/NAD+ ratios than complexes isolated from aerobic organisms, as would be predicted for PDCs from organisms exploiting microaerobic habitats. These results suggest that although these helminths have similar anaerobic mitochondrial pathways, they contain significantly different PDCs.

Changing phenotypic expression in a patient with a mitochondrial encephalopathy due to 13042G>A de novo mutation--a 5 year follow up.

PubMed

Schinwelski, M; Kierdaszuk, B; Dulski, J; Tońska, K; Kodroń, A; Sitek, E J; Bartnik, E; Kamińska, A; Kwieciński, H; Sławek, J

2015-08-01

Mutations in NADH dehydrogenase (ND) subunits of complex I lead to mitochondrial encephalomyopathies associated with various phenotypes. This report aims to present the patient's clinical symptomatology in the context of a very rare 13042G>A de novo mutation and with an emphasis on changing phenotypic expression and pronounced, long-standing response to levetiracetam.

Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase

PubMed Central

Tuz, Karina; Li, Chen; Fang, Xuan; Raba, Daniel A.; Liang, Pingdong; Minh, David D. L.; Juárez, Oscar

2017-01-01

The sodium-dependent NADH dehydrogenase (Na+-NQR) is a key component of the respiratory chain of diverse prokaryotic species, including pathogenic bacteria. Na+-NQR uses the energy released by electron transfer between NADH and ubiquinone (UQ) to pump sodium, producing a gradient that sustains many essential homeostatic processes as well as virulence factor secretion and the elimination of drugs. The location of the UQ binding site has been controversial, with two main hypotheses that suggest that this site could be located in the cytosolic subunit A or in the membrane-bound subunit B. In this work, we performed alanine scanning mutagenesis of aromatic residues located in transmembrane helices II, IV, and V of subunit B, near glycine residues 140 and 141. These two critical glycine residues form part of the structures that regulate the site's accessibility. Our results indicate that the elimination of phenylalanine residue 211 or 213 abolishes the UQ-dependent activity, produces a leak of electrons to oxygen, and completely blocks the binding of UQ and the inhibitor HQNO. Molecular docking calculations predict that UQ interacts with phenylalanine 211 and pinpoints the location of the binding site in the interface of subunits B and D. The mutagenesis and structural analysis allow us to propose a novel UQ-binding motif, which is completely different compared with the sites of other respiratory photosynthetic complexes. These results are essential to understanding the electron transfer pathways and mechanism of Na+-NQR catalysis. PMID:28053088

Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.

PubMed

Soreze, Yohan; Boutron, Audrey; Habarou, Florence; Barnerias, Christine; Nonnenmacher, Luc; Delpech, Hélène; Mamoune, Asmaa; Chrétien, Dominique; Hubert, Laurence; Bole-Feysot, Christine; Nitschke, Patrick; Correia, Isabelle; Sardet, Claude; Boddaert, Nathalie; Hamel, Yamina; Delahodde, Agnès; Ottolenghi, Chris; de Lonlay, Pascale

2013-12-17

Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes. Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and α-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts. Exome sequencing identified two heterozygous mutations (c.875C > G and c.535A > G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, α-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and α-KGDH E2 subunits. Accordingly, the production of 14CO2 by patient fibroblasts after incubation with 14Cglucose, 14Cbutyrate or 14C3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and α-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants. We report here a putative case of impaired free or H protein-derived lipoic acid attachment due to LIPT1 mutations as a cause of PDH and α-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies.

Distribution of the branched-chain α-ketoacid dehydrogenase complex E1α subunit and glutamate dehydrogenase in the human brain and their role in neuro-metabolism.

PubMed

Hull, Jonathon; Usmari Moraes, Marcela; Brookes, Emma; Love, Seth; Conway, Myra E

2018-01-01

Glutamate is the major excitatory neurotransmitter of the central nervous system, with the branched-chain amino acids (BCAAs) acting as key nitrogen donors for de novo glutamate synthesis. Despite the importance of these major metabolites, their metabolic pathway in the human brain is still not well characterised. The metabolic pathways that influence the metabolism of BCAAs have been well characterised in rat models. However, the expression of key proteins such as the branched-chain α-ketoacid dehydrogenase (BCKD) complex and glutamate dehydrogenase isozymes (GDH) in the human brain is still not well characterised. We have used specific antibodies to these proteins to analyse their distribution within the human brain and report, for the first time, that the E1α subunit of the BCKD is located in both neurons and vascular endothelial cells. We also demonstrate that GDH is localised to astrocytes, although vascular immunolabelling does occur. The labelling of GDH was most intense in astrocytes adjacent to the hippocampus, in keeping with glutamatergic neurotransmission in this region. GDH was also present in astrocyte processes abutting vascular endothelial cells. Previously, we demonstrated that the branched-chain aminotransferase (hBCAT) proteins were most abundant in vascular cells (hBCATm) and neurons (hBCATc). Present findings are further evidence that BCAAs are metabolised within both the vasculature and neurons in the human brain. We suggest that GDH, hBCAT and the BCKD proteins operate in conjunction with astrocytic glutamate transporters and glutamine synthetase to regulate the availability of glutamate. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

PubMed Central

Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy

2014-01-01

Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization. PMID:24852961

Counseling patients with succinate dehydrogenase subunit defects: genetics, preventive guidelines, and dealing with uncertainty

PubMed Central

Raygada, Margarita; King, Kathryn S.; Adams, Karen T.; Stratakis, Constantine A.; Pacak, Karel

2016-01-01

The discovery that mutations in the succinate dehydrogenase (SDH) complex subunit (SDHA, B/C/D/AF2) genes predispose patients to the development of tumors has led to the identification of a large population of patients and relatives at risk for developing malignancies. The most frequent conditions associated with these mutations are the familial paraganglioma syndromes. Other tumors that are frequently associated with SDH mutations (SDHx) are gastrointestinal stromal tumors and renal cell carcinomas. A number of other rare associations have also been described. SDHx mutations are often clinically silent and metastatic, but they may also be aggressive in their presentation. The penetrance of these mutations is beginning to be understood, and the characteristics of the phenotype are being elucidated. However, the inability to accurately predict the appearance, nature, and location of tumors as well as their tendency to recur or metastasize pose challenges to those who counsel and manage patients with SDHx mutations. In this work, we present our approach for counseling these families in the context of the current uncertainties, while striving to maintain patient autonomy. PMID:24854530

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

PubMed

Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Lu, Jie; Maringer, Katherine V; Sims-Lucas, Sunder; Prochownik, Edward V; Gibson, Bradford W; Goetzman, Eric S

2017-06-16

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Leigh disease presenting in utero due to a novel missense mutation in the mitochondrial DNA-ND3.

PubMed

Leshinsky-Silver, Esther; Lev, Dorit; Malinger, Gustavo; Shapira, Daniel; Cohen, Sarit; Lerman-Sagie, Tally; Saada, Ann

2010-05-01

Leigh syndrome can be caused by defects in both nuclear and mitochondrial genes involved in energy metabolism. Recently, an increasing number of mutations in mitochondrial DNA encoding regions, especially in NADH dehydrogenase (respiratory chain complex I) subunits, have been reported as causative of early onset Leigh syndrome. We describe a patient whose fetal brain ultrasound demonstrated periventricular pseudocyst suggestive of a possible mitochondrial disorder who presented postnatally with Leigh syndrome. A muscle biopsy demonstrated a partial decrease in complex I and pyruvate dehydrogenase (PDH-E1 alpha) activity. Sequencing of the PDH-E1 alpha gene did not reveal any mutation. Sequencing of the mtDNA revealed a novel heteroplasmic G10254A (D66N) mutation in the ND3 gene. This change results in a substitution of aspartic acid to asparagine in a highly conserved domain of the ND3 subunit. The mutation could not be detected in the mother's blood or urine sediment. Blue native gel electrophoresis of muscle mitochondria revealed a normal size, albeit a decreased level of complex I. The G10254A substitution in the mtDNA-ND3 gene is another cause of maternally inherited Leigh syndrome. This case demonstrates that periventricular pseudocysts may be the initial in utero presentation in patients with mitochondrial disorders. We emphasize the importance of screening the mtDNA in pediatric patients as the first step in molecular diagnosis of Leigh syndrome. (c) 2010 Elsevier Inc. All rights reserved.

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

2009-06-08

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips tomore » the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Desai, S.; Ruff, V.; DuBrul, E.F.

The pyruvate dehydrogenase complex (PDC) plays a pivotal role in the anaerobic metabolism of Ascaris suum mitochondria. They have initiated a series of studies on the in vitro synthesis and mitochondrial import of PDC. PDC has been purified from adult Ascaris body wall muscle, fully phosphorylated in vitro, and separated into its component subunits on SDS/PAGE. The individual components were electroeluted from the gels and used to immunize rabbits. IgG's to the individual subunits were prepared from antisera and their specificities were verified by immuno-blotting. Each IgG identified a single specific band at the appropriate location in extracts of adultmore » Ascaris body wall muscle mitochondria. Poly A/sup +/-RNA was prepared from body wall muscle and translated in a reticylocyte lysate system using /sup 35/S-methionine. Translation products were immunoprecipitated with specific IgG's, electrophoresed, and fluorographed. Each immunoprecipitation gave rise to a single radioactive polypeptide that was slightly larger than the specific PDC subunit isolated from the adult mitochondria. This system has demonstrated its feasibility for the study of mitochondrial import of a multienzyme complex that is critical for the anaerobic mitochondrial metabolism of Ascaris suum.« less

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells.

PubMed

Okada, M; Shimura, K; Shiraki, H; Nakagawa, H

1983-11-01

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malik, Radhika; Viola, Ronald E.

2010-10-28

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identificationmore » of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.« less

XoxF Is Required for Expression of Methanol Dehydrogenase in Methylobacterium extorquens AM1 ▿

PubMed Central

Skovran, Elizabeth; Palmer, Alexander D.; Rountree, Austin M.; Good, Nathan M.; Lidstrom, Mary E.

2011-01-01

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes. PMID:21873495

Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

PubMed Central

Burdette, D; Zeikus, J G

1994-01-01

The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

PubMed Central

Kozak, Barbara U.; van Rossum, Harmen M.; Luttik, Marijke A. H.; Akeroyd, Michiel; Benjamin, Kirsten R.; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T.

2014-01-01

ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. PMID:25336454

Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

PubMed

Schmiesing, Jessica; Schlüter, Hartmut; Ullrich, Kurt; Braulke, Thomas; Mühlhausen, Chris

2014-01-01

Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST) involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB) serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

Interaction of Glutaric Aciduria Type 1-Related glutaryl-CoA Dehydrogenase with Mitochondrial Matrix Proteins

PubMed Central

Schmiesing, Jessica; Schlüter, Hartmut; Ullrich, Kurt; Braulke, Thomas; Mühlhausen, Chris

2014-01-01

Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST) involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB) serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1. PMID:24498361

Mediated Plastid RNA Editing in Plant Immunity

PubMed Central

García-Andrade, Javier; Ramírez, Vicente; López, Ana; Vera, Pablo

2013-01-01

Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes. PMID:24204264

Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer.

PubMed

Stacpoole, Peter W

2017-11-01

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Function of Several Critical Amino Acids in Human Pyruvate Dehydrogenase Revealed by Its Structure

NASA Technical Reports Server (NTRS)

Korotchkina, Lioubov G.; Ciszak, E.; Patel, M.

2004-01-01

Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of beta W135, alpha P188, alpha M181, alpha H15 and alpha R349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: beta W135 is the center residue in the hydrophobic interaction between beta and beta' subunits; alpha P188 and alpha M181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alpha H15, is necessary for the organization of the N-terminus of alpha and alpha'; subunits and alpha R349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

Pyruvate dehydrogenase deficiency and epilepsy.

PubMed

Prasad, Chitra; Rupar, Tony; Prasad, Asuri N

2011-11-01

The pyruvate dehydrogenase complex (PDHc) is a mitochondrial matrix multienzyme complex that provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the conversion of pyruvate into acetyl-CoA. PDHc deficiency is one of the commoner metabolic disorders of lactic acidosis presenting with neurological phenotypes that vary with age and gender. In this mini-review, we postulate mechanisms of epilepsy in the setting of PDHc deficiency using two illustrative cases (one with pyruvate dehydrogenase complex E1-alpha polypeptide (PDHA1) deficiency and the second one with pyruvate dehydrogenase complex E1-beta subunit (PDHB) deficiency (a rare subtype of PDHc deficiency)) and a selected review of published case series. PDHc plays a critical role in the pathway of carbohydrate metabolism and energy production. In severe deficiency states the resulting energy deficit impacts on brain development in utero resulting in structural brain anomalies and epilepsy. Milder deficiency states present with variable manifestations that include cognitive delay, ataxia, and seizures. Epileptogenesis in PDHc deficiency is linked to energy failure, development of structural brain anomalies and abnormal neurotransmitter metabolism. The use of the ketogenic diet bypasses the metabolic block, by providing a direct source of acetyl-CoA, leading to amelioration of some symptoms. Genetic counseling is essential as PDHA1 deficiency (commonest defect) is X-linked although females can be affected due to unfavorable lyonization, while PDHB and PDH phosphatase (PDP) deficiencies (much rarer defects) are of autosomal recessive inheritance. Research is in progress for looking into animal models to better understand pathogenesis and management of this challenging disorder. Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in Syntrophomonas wolfei

PubMed Central

Schmidt, Alexander; Müller, Nicolai; Schink, Bernhard; Schleheck, David

2013-01-01

In syntrophic conversion of butyrate to methane and CO2, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO2 reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate. In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases. Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation. PMID:23468890

Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

DOE PAGES

Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; ...

2015-04-21

Inosine 5´-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH ( CpIMPDH) in complex with inosine 5´-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO 2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is amore » new strategy for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications.« less

Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar

2015-04-21

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment ofCryptosporidiuminfections. Here, the structure ofC. parvumIMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor within vivoanticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO 2moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization ofC. parvuminhibitorsmore » for both antiparasitic and antibacterial applications.« less

Pyruvate dehydrogenase complex deficiency and its relationship with epilepsy frequency--An overview.

PubMed

Bhandary, Suman; Aguan, Kripamoy

2015-10-01

The pyruvate dehydrogenase complex (PDHc) is a member of a family of multienzyme complexes that provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the physiologically irreversible decarboxylation of various 2-oxoacid substrates to their corresponding acyl-CoA derivatives, NADH and CO2. PDHc deficiency is a metabolic disorder commonly associated with lactic acidosis, progressive neurological and neuromuscular degeneration that vary with age and gender. In this review, we aim to discuss the relationship between occurrence of epilepsy and PDHc deficiency associated with the pyruvate dehydrogenase complex (E1α subunit (PDHA1) and E1β subunit (PDHB)) and PDH phosphatase (PDP) deficiency. PDHc plays a crucial role in the aerobic carbohydrate metabolism and regulates the use of carbohydrate as the source of oxidative energy. In severe PDHc deficiency, the energy deficit impairs brain development in utero resulting in physiological and structural changes in the brain that contributes to the subsequent onset of epileptogenesis. Epileptogenesis in PDHc deficiency is linked to energy failure and abnormal neurotransmitter metabolism that progressively alters neuronal excitability. This metabolic blockage might be restricted via inclusion of ketogenic diet that is broken up by β-oxidation and directly converting it to acetyl-CoA, and thereby improving the patient's health condition. Genetic counseling is essential as PDHA1 deficiency is X-linked. The demonstration of the X-chromosome localization of PDHA1 resolved a number of questions concerning the variable phenotype displayed by patients with E1 deficiency. Most patients show a broad range of neurological abnormalities, with the severity showing some dependence on the nature of the mutation in the Elα gene, while PDHB and PDH phosphatase (PDP) deficiencies are of autosomal recessive inheritance. However, in females, the disorder is further complicated by the pattern of X-chromosome inactivation, i.e., unfavorable lyonization. Furthermore research should focus on epileptogenic animal models; this might pave a new way toward identification of the pathophysiology of this challenging disorder. Copyright © 2015 Elsevier B.V. All rights reserved.

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

PubMed Central

Chen, Chwen-Lih; Zhang, Liwen; Yeh, Alexander; Chen, Chun-An; Green-Church, Kari B.; Zweier, Jay L.; Chen, Yeong-Renn

2008-01-01

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2·-) and O2·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2·- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool. PMID:17444656

The electrochemical behavior of a FAD dependent glucose dehydrogenase with direct electron transfer subunit by immobilization on self-assembled monolayers.

PubMed

Lee, Inyoung; Loew, Noya; Tsugawa, Wakako; Lin, Chi-En; Probst, David; La Belle, Jeffrey T; Sode, Koji

2018-06-01

Continuous glucose monitoring (CGM) is a vital technology for diabetes patients by providing tight glycemic control. Currently, many commercially available CGM sensors use glucose oxidase (GOD) as sensor element, but this enzyme is not able to transfer electrons directly to the electrode without oxygen or an electronic mediator. We previously reported a mutated FAD dependent glucose dehydrogenase complex (FADGDH) capable of direct electron transfer (DET) via an electron transfer subunit without involving oxygen or a mediator. In this study, we investigated the electrochemical response of DET by controlling the immobilization of DET-FADGDH using 3 types of self-assembled monolayers (SAMs) with varying lengths. With the employment of DET-FADGDH and SAM, high current densities were achieved without being affected by interfering substances such as acetaminophen and ascorbic acid. Additionally, the current generated from DET-FADGDH electrodes decreased with increasing length of SAM, suggesting that the DET ability can be affected by the distance between the enzyme and the electrode. These results indicate the feasibility of controlling the immobilization state of the enzymes on the electrode surface. Copyright © 2017. Published by Elsevier B.V.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nikolau, Basil J; Wurtele, Eve S; Oliver, David J

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method ofmore » producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.« less

Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase

PubMed Central

2013-01-01

Background Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes. Methods Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and α-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts. Results Exome sequencing identified two heterozygous mutations (c.875C > G and c.535A > G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, α-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and α-KGDH E2 subunits. Accordingly, the production of 14CO2 by patient fibroblasts after incubation with 14Cglucose, 14Cbutyrate or 14C3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and α-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants. Conclusion We report here a putative case of impaired free or H protein-derived lipoic acid attachment due to LIPT1 mutations as a cause of PDH and α-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies. PMID:24341803

TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

PubMed

Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2014-03-01

Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes.

PubMed

Caballero, Susana; Duchêne, Sebastian; Garavito, Manuel F; Slikas, Beth; Baker, C Scott

2015-01-01

A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the 'river dolphins', early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three 'river dolphins' (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.

Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes

PubMed Central

Caballero, Susana; Duchêne, Sebastian; Garavito, Manuel F.; Slikas, Beth; Baker, C. Scott

2015-01-01

A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the ‘river dolphins’, early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three ‘river dolphins’ (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans. PMID:25946045

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

PubMed

Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

2008-09-15

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

DOEpatents

Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

2005-09-13

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method ofmore » producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.« less

The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation

PubMed Central

Kiss, Gergely; Konrad, Csaba; Doczi, Judit; Starkov, Anatoly A.; Kawamata, Hibiki; Manfredi, Giovanni; Zhang, Steven F.; Gibson, Gary E.; Beal, M. Flint; Adam-Vizi, Vera; Chinopoulos, Christos

2013-01-01

A decline in α-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20–48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ∼30% higher ADP-ATP exchange rates compared to those obtained from DLST+/− or DLD+/− littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on “in-house” mitochondrial ATP reserves.—Kiss, G., Konrad, C., Doczi, J., Starkov, A. A., Kawamata, H., Manfredi, G., Zhang, S. F., Gibson, G. E., Beal, M. F., Adam-Vizi, V., Chinopoulos, C. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation. PMID:23475850

The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PubMed

Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

2016-06-01

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

PubMed

Mkrtchyan, Garik V; Üçal, Muammer; Müllebner, Andrea; Dumitrescu, Sergiu; Kames, Martina; Moldzio, Rudolf; Molcanyi, Marek; Schaefer, Samuel; Weidinger, Adelheid; Schaefer, Ute; Hescheler, Juergen; Duvigneau, Johanna Catharina; Redl, Heinz; Bunik, Victoria I; Kozlov, Andrey V

2018-05-16

Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings. Copyright © 2018. Published by Elsevier B.V.

Structural Studies of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

2002-01-01

Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

PubMed

Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

2015-05-04

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain.

PubMed

Mu, Hong-Na; Li, Quan; Pan, Chun-Shui; Liu, Yu-Ying; Yan, Li; Hu, Bai-He; Sun, Kai; Chang, Xin; Zhao, Xin-Rong; Fan, Jing-Yu; Han, Jing-Yan

2015-08-01

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain. Copyright © 2015 Elsevier Inc. All rights reserved.

Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

PubMed

Geiss, K T; Abbas, G M; Makaroff, C A

1994-04-01

The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

Lipoic acid biosynthesis defects.

PubMed

Mayr, Johannes A; Feichtinger, René G; Tort, Frederic; Ribes, Antonia; Sperl, Wolfgang

2014-07-01

Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

PubMed Central

Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

2014-01-01

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

PubMed Central

Hensgens, C M; Vonck, J; Van Beeumen, J; van Bruggen, E F; Hansen, T A

1993-01-01

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH. Images PMID:8491707

A large deletion in the succinate dehydrogenase B gene (SDHB) in a Japanese patient with abdominal paraganglioma and concomitant metastasis.

PubMed

Kodama, Hitomi; Iihara, Masatoshi; Nissato, Sumiko; Isobe, Kazumasa; Kawakami, Yasushi; Okamoto, Takahiro; Takekoshi, Kazuhiro

2010-01-01

Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.

The mitochondrial electron transfer flavoprotein complex is essential for survival of Arabidopsis in extended darkness.

PubMed

Ishizaki, Kimitsune; Schauer, Nicolas; Larson, Tony R; Graham, Ian A; Fernie, Alisdair R; Leaver, Christopher J

2006-09-01

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.

Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wynn, R. Max; Kato, Masato; Chuang, Jacinta L.

2008-10-21

Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-{angstrom} crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, comparedmore » with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp{sup 394}-Trp{sup 395}) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.« less

Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity.

PubMed

Wynn, R Max; Kato, Masato; Chuang, Jacinta L; Tso, Shih-Chia; Li, Jun; Chuang, David T

2008-09-12

Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

PubMed Central

Lloyd, Julie C.; Raines, Christine A.

2011-01-01

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. PMID:21498632

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

PubMed

Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

2011-07-01

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

PubMed

Fontaine, Jean-Xavier; Saladino, Francesca; Agrimonti, Caterina; Bedu, Magali; Tercé-Laforgue, Thérèse; Tétu, Thierry; Hirel, Bertrand; Restivo, Francesco M; Dubois, Frédéric

2006-03-01

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes.

PubMed

Xiao, Wusheng; Sarsour, Ehab H; Wagner, Brett A; Doskey, Claire M; Buettner, Garry R; Domann, Frederick E; Goswami, Prabhat C

2016-02-01

Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of 4-monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ-treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ-induced toxicity.

Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

PubMed

Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

2014-10-21

The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals. Copyright © 2014 Kozak et al.

Enhanced expression of the Escherichia coli serA gene in a plasmid vector. Purification, crystallization, and preliminary X-ray data of D-3 phosphoglycerate dehydrogenase.

PubMed

Schuller, D J; Fetter, C H; Banaszak, L J; Grant, G A

1989-02-15

The serA gene of Escherichia coli strain K-12, which codes for the cooperative allosteric enzyme D-3-phosphoglycerate dehydrogenase, was inserted into an inducible expression vector which produced phosphoglycerate dehydrogenase as 8% of the soluble protein of E. coli. The purified protein was used to grow several different single crystal forms. One of these, with space group P2(1), appears to contain all four subunits of the tetrameric enzyme in the asymmetric unit and diffracts to sufficient resolution to allow determination of the structure of phosphoglycerate dehydrogenase.

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

PubMed

Rurek, Michal; Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2015-01-01

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular characterization of maple syrup urine disease patients from Tunisia.

PubMed

Jaafar, N; Moleirinho, A; Kerkeni, E; Monastiri, K; Seboui, H; Amorim, A; Prata, M J; Quental, S

2013-03-15

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1β and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene. Copyright © 2012 Elsevier B.V. All rights reserved.

Leigh syndrome caused by mutations in the flavoprotein (Fp) subunit of succinate dehydrogenase (SDHA).

PubMed

Horváth, R; Abicht, A; Holinski-Feder, E; Laner, A; Gempel, K; Prokisch, H; Lochmüller, H; Klopstock, T; Jaksch, M

2006-01-01

Detailed clinical, neuroradiological, histological, biochemical, and genetic investigations were undertaken in a child suffering from Leigh syndrome. The clinical symptoms started at age five months and led to a severe progressive neurodegenerative disorder causing epilepsy, psychomotor retardation, and tetraspasticity. Biochemical measurement of skeletal muscle showed a severe decrease in mitochondrial complex II. Sequencing of SDHA revealed compound heterozygosity for a nonsense mutation in exon 4 (W119X) and a missense mutation in exon 3 (A83V), both absent in normal controls. In six additional patients--five with Leigh or Leigh-like syndrome and one with neuropathy and ataxia associated with isolated deficiency of complex II--mutations in SDHA were not detected, indicating genetic heterogeneity.

Cardiolipin deficiency causes a dissociation of the b 6 c:caa 3 megacomplex in B. subtilis membranes.

PubMed

García Montes de Oca, Led Yered Jafet; Cabellos Avelar, Tecilli; Picón Garrido, Gerardo Ignacio; Chagoya-López, Alicia; González de la Vara, Luis; Delgado Buenrostro, Norma Laura; Chirino-López, Yolanda Irasema; Gómez-Lojero, Carlos; Gutiérrez-Cirlos, Emma Berta

2016-08-01

The associations among respiratory complexes in energy-transducing membranes have been established. In fact, it is known that the Gram-negative bacteria Paracoccus denitrificans and Escherichia coli have respiratory supercomplexes in their membranes. These supercomplexes are important for channeling substrates between enzymes in a metabolic pathway, and the assembly of these supercomplexes depends on the protein subunits and membrane lipids, mainly cardiolipin, which is present in both the mitochondrial inner membrane and bacterial membranes. The Gram-positive bacterium Bacillus subtilis has a branched respiratory chain, in which some complexes generate proton motive force whereas others constitute an escape valve of excess reducing power. Some peculiarities of this respiratory chain are the following: a type II NADH dehydrogenase, a unique b 6 c complex that has a b 6 type cytochrome with a covalently bound heme, and a c-type heme attached to the third subunit, which is similar to subunit IV of the photosynthetic b 6 f complex. Cytochrome c oxygen reductase (caa 3 ) contains a c-type cytochrome on subunit I. We previously showed that the b 6 c and the caa 3 complexes form a supercomplex. Both the b 6 c and the caa 3 together with the quinol oxygen reductase aa 3 generate the proton motive force in B. subtilis. In order to seek proof that this supercomplex is important for bacterial growth in aerobic conditions we compared the b 6 c: caa 3 supercomplex from wild type membranes with membranes from two mutants lacking cardiolipin. Both mutant complexes were found to have similar activity and heme content as the wild type. Clear native electrophoresis showed that mutants lacking cardiolipin had b 6 c:caa 3 supercomplexes of lower mass or even individual complexes after membrane solubilization with digitonin. The use of dodecyl maltoside revealed a more evident difference between wild-type and mutant supercomplexes. Here we provide evidence showing that cardiolipin plays a role in the stability of the b 6 c:caa 3 supercomplex in B. subtilis.

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

PubMed

Uttaro, A D; Opperdoes, F R

1997-04-01

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid–Mediated Effect on Stomatal Aperture[W][OA

PubMed Central

Araújo, Wagner L.; Nunes-Nesi, Adriano; Osorio, Sonia; Usadel, Björn; Fuentes, Daniela; Nagy, Réka; Balbo, Ilse; Lehmann, Martin; Studart-Witkowski, Claudia; Tohge, Takayuki; Martinoia, Enrico; Jordana, Xavier; DaMatta, Fábio M.; Fernie, Alisdair R.

2011-01-01

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell–specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture. PMID:21307286

Inhibition of Pyruvate Dehydrogenase Kinase as a Therapeutic Strategy against Cancer.

PubMed

Sradhanjali, Swatishree; Reddy, Mamatha M

2018-05-22

Cancer cells alter their metabolism to support the uninterrupted supply of biosynthetic molecules required for continuous proliferation. Glucose metabolism is frequently reprogrammed in several tumors in addition to fatty acid, amino acid and glutamine metabolism. Pyruvate dehydrogenase kinase (PDK) is a gatekeeper enzyme involved in altered glucose metabolism in tumors. There are four isoforms of PDK (1 to 4) in humans. PDK phosphorylates E1α subunit of pyruvate dehydrogenase complex (PDC) and inactivates it. PDC decarboxylates pyruvate to acetyl CoA, which is further metabolized in mitochondria. Overexpression of PDK was observed in several tumors and is frequently associated with chemotherapy related drug resistance, invasion and metastasis. Elevated expression of PDK leads to a shift in glucose metabolism towards glycolysis instead of oxidative phosphorylation. This review summarizes recent literature related to the role of PDKs in cancer and their inhibition as a strategy. In particular, we discuss the role of PDK in tumor progression, metabolic reprogramming in stem cells, and their regulation by miRNAs and lncRNAs, oncogenes and tumor suppressors. Further, we review strategies aimed at targeting PDK to halt tumor growth and progression. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

PubMed Central

Negi, Surendra S.; Carol, Andrew A.; Pandya, Shivangi; Braun, Werner; Anderson, Louise E.

2008-01-01

In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex. PMID:17945509

Distinct symmetry and limited peptide refolding activity of the thermosomes from the acidothermophilic archaea Acidianus tengchongensis S5{sup T}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Li; Hu, Zhong-jun; Luo, Yuan-ming

Recombinant thermosomes from the Acidianus tengchongensis strain S5{sup T} were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpn{alpha} or rATcpn{beta}) and hetero-oligomers (rATcpn{alpha}{beta}). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpn{alpha} homo-oligomer was shown to possess 8-fold symmetry while both rATcpn{beta} and rATcpn{alpha}{beta} oligomers adopted 9-fold symmetry. rATcpn{alpha}{beta} oligomers were shown to contain the {alpha} and {beta} subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 {sup o}C and completely prevented the formation of aggregates during thermal inactivation of citrate synthasemore » at 45 {sup o}C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpn{beta} sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.« less

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe.

PubMed

Maclean, Andrew E; Hertle, Alexander P; Ligas, Joanna; Bock, Ralph; Balk, Janneke; Meyer, Etienne H

2018-05-21

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I. Copyright © 2018 Elsevier Ltd. All rights reserved.

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

PubMed Central

Steenkamp, D J

1987-01-01

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA. Images Fig. 2. Fig. 3. Fig. 5. PMID:3115254

Cancer-associated Isocitrate Dehydrogenase Mutations Inactivate NADPH-dependent Reductive Carboxylation*

PubMed Central

Leonardi, Roberta; Subramanian, Chitra; Jackowski, Suzanne; Rock, Charles O.

2012-01-01

Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO2-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP+ and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired. PMID:22442146

HIV-1 Vpr Enhances PPARβ/δ-Mediated Transcription, Increases PDK4 Expression, and Reduces PDC Activity

PubMed Central

Shrivastav, Shashi; Zhang, Liyan; Okamoto, Koji; Lee, Hewang; Lagranha, Claudia; Abe, Yoshifusa; Balasubramanyam, Ashok; Lopaschuk, Gary D.; Kino, Tomoshige

2013-01-01

HIV infection and its therapy are associated with disorders of lipid metabolism and bioenergetics. Previous work has suggested that viral protein R (Vpr) may contribute to the development of lipodystrophy and insulin resistance observed in HIV-1–infected patients. In adipocytes, Vpr suppresses mRNA expression of peroxisomal proliferator-activating receptor-γ (PPARγ)-responsive genes and inhibits differentiation. We investigated whether Vpr might interact with PPARβ/δ and influence its transcriptional activity. In the presence of PPARβ/δ, Vpr induced a 3.3-fold increase in PPAR response element-driven transcriptional activity, a 1.9-fold increase in pyruvate dehydrogenase kinase 4 (PDK4) protein expression, and a 1.6-fold increase in the phosphorylated pyruvate dehydrogenase subunit E1α leading to a 47% decrease in the activity of the pyruvate dehydrogenase complex in HepG2 cells. PPARβ/δ knockdown attenuated Vpr-induced enhancement of endogenous PPARβ/δ-responsive PDK4 mRNA expression. Vpr induced a 1.3-fold increase in mRNA expression of both carnitine palmitoyltransferase I (CPT1) and acetyl-coenzyme A acyltransferase 2 (ACAA2) and doubled the activity of β-hydroxylacyl coenzyme A dehydrogenase (HADH). Vpr physically interacted with the ligand-binding domain of PPARβ/δ in vitro and in vivo. Consistent with a role in energy expenditure, Vpr increased state-3 respiration in isolated mitochondria (1.16-fold) and basal oxygen consumption rate in intact HepG2 cells (1.2-fold) in an etomoxir-sensitive manner, indicating that the oxygen consumption rate increase is β-oxidation–dependent. The effects of Vpr on PPAR response element activation, pyruvate dehydrogenase complex activity, and β-oxidation were reversed by specific PPARβ/δ antagonists. These results support the hypothesis that Vpr contributes to impaired energy metabolism and increased energy expenditure in HIV patients. PMID:23842279

Novel functions of the α-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer’s disease

PubMed Central

Shi, Qingli; Xu, Hui; Kleinman, Wayne A.; Gibson, Gary E.

2011-01-01

Measures in autopsied brains from Alzheimer’s Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H2O2 and to probe the mechanism underlying these changes. Since the response to H2O2 is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one hour treatment with H2O2 decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H2O2 inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H2O2 converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H2O2 diminished glutathione to a similar level after 24 hrs. However, H2O2, but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H2O2 together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes. PMID:18206986

Antisense Inhibition of the 2-Oxoglutarate Dehydrogenase Complex in Tomato Demonstrates Its Importance for Plant Respiration and during Leaf Senescence and Fruit Maturation[W][OA

PubMed Central

Araújo, Wagner L.; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Nunes-Nesi, Adriano; Fernie, Alisdair R.

2012-01-01

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon–nitrogen interactions. PMID:22751214

Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

PubMed Central

Ferriero, Rosa; Manco, Giuseppe; Lamantea, Eleonora; Nusco, Edoardo; Ferrante, Mariella I.; Sordino, Paolo; Stacpoole, Peter W.; Lee, Brendan; Zeviani, Massimo; Brunetti-Pierri, Nicola

2014-01-01

Lactic acidosis is a build-up of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57B6/L wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noam631 zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis. PMID:23467562

The β and γ subunits play distinct functional roles in the α2βγ heterotetramer of human NAD-dependent isocitrate dehydrogenase

NASA Astrophysics Data System (ADS)

Ma, Tengfei; Peng, Yingjie; Huang, Wei; Liu, Yabing; Ding, Jianping

2017-01-01

Human NAD-dependent isocitrate dehydrogenase existing as the α2βγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory β and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αβ and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of α2βγ requires the assembly and cooperative function of both heterodimers. α2βγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S0.5,isocitrate and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate α2βγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αβ. Furthermore, the allosteric activation of α2βγ is through the γ subunit not the β subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme.

BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei

PubMed Central

Oppenheim, Rebecca D.; Limenitakis, Julien; Polonais, Valerie; Seeber, Frank; Barrett, Michael P.; Billker, Oliver; McConville, Malcolm J.; Soldati-Favre, Dominique

2014-01-01

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens. PMID:25032958

Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108.

PubMed

Yakushi, Toshiharu; Fukunari, Seiya; Kodama, Tomohiro; Matsutani, Minenosuke; Nina, Shun; Kataoka, Naoya; Theeragool, Gunjana; Matsushita, Kazunobu

2018-05-01

Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes-alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)-of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The ΔaldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact ΔaldH cells and the ΔaldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent K M values of the wild-type and ΔaldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where ΔaldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent K M value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

PubMed

Hiller, Sylvia; DeKroon, Robert; Hamlett, Eric D; Xu, Longquan; Osorio, Cristina; Robinette, Jennifer; Winnik, Witold; Simington, Stephen; Maeda, Nobuyo; Alzate, Oscar; Yi, Xianwen

2016-01-01

S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

PubMed Central

Roberts, David L.; Frerman, Frank E.; Kim, Jung-Ja P.

1996-01-01

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins. PMID:8962055

Identification of a Dehydrogenase Required for Lactose Metabolism in Caulobacter crescentus▿ †‡

PubMed Central

Arellano, Benjamin H.; Ortiz, Janett D.; Manzano, Janet; Chen, Joseph C.

2010-01-01

Caulobacter crescentus, which thrives in freshwater environments with low nutrient levels, serves as a model system for studying bacterial cell cycle regulation and organelle development. We examined its ability to utilize lactose (i) to gain insight into the metabolic capacities of oligotrophic bacteria and (ii) to obtain an additional genetic tool for studying this model organism, aiming to eliminate the basal enzymatic activity that hydrolyzes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Using a previously isolated transposon mutant, we identified a gene, lacA, that is required for growth on lactose as the sole carbon source and for turning colonies blue in the presence of X-gal. LacA, which contains a glucose-methanol-choline (GMC) oxidoreductase domain, has homology to the flavin subunit of Pectobacterium cypripedii's gluconate dehydrogenase. Sequence comparisons indicated that two genes near lacA, lacB and lacC, encode the other subunits of the membrane-bound dehydrogenase. In addition to lactose, all three lac genes are involved in the catabolism of three other β-galactosides (lactulose, lactitol, and methyl-β-d-galactoside) and two glucosides (salicin and trehalose). Dehydrogenase assays confirmed that the lac gene products oxidize lactose, salicin, and trehalose. This enzymatic activity is inducible, and increased lac expression in the presence of lactose and salicin likely contributes to the induction. Expression of lacA also depends on the presence of the lac genes, implying that the dehydrogenase participates in induction. The involvement of a dehydrogenase suggests that degradation of lactose and other sugars in C. crescentus may resemble a proposed pathway in Agrobacterium tumefaciens. PMID:20190087

An evolutionary switch in ND2 enables Src kinase regulation of NMDA receptors

NASA Astrophysics Data System (ADS)

Scanlon, David P.; Bah, Alaji; Krzeminski, Mickaël; Zhang, Wenbo; Leduc-Pessah, Heather L.; Dong, Yi Na; Forman-Kay, Julie D.; Salter, Michael W.

2017-05-01

The non-receptor tyrosine kinase Src is a key signalling hub for upregulating the function of N-methyl D-aspartate receptors (NMDARs). Src is anchored within the NMDAR complex via NADH dehydrogenase subunit 2 (ND2), a mitochondrially encoded adaptor protein. The interacting regions between Src and ND2 have been broadly identified, but the interaction between ND2 and the NMDAR has remained elusive. Here we generate a homology model of ND2 and dock it onto the NMDAR via the transmembrane domain of GluN1. This interaction is enabled by the evolutionary loss of three helices in bilaterian ND2 proteins compared to their ancestral homologues. We experimentally validate our model and demonstrate that blocking this interaction with an ND2 fragment identified in our experimental studies prevents Src-mediated upregulation of NMDAR currents in neurons. Our findings establish the mode of interaction between an NMDAR accessory protein with one of the core subunits of the receptor.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

PubMed

Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

2010-08-01

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species.

PubMed

Droux, M; Jacquot, J P; Miginac-Maslow, M; Gadal, P; Huet, J C; Crawford, N A; Yee, B C; Buchanan, B B

1987-02-01

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

Proteomics of a new esophageal cancer cell line established from Persian patient.

PubMed

Moghanibashi, Mehdi; Jazii, Ferdous Rastgar; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2012-05-25

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study. Copyright © 2012 Elsevier B.V. All rights reserved.

Cloning and polymorphisms of yak lactate dehydrogenase B gene.

PubMed

Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

2013-06-05

The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

PubMed Central

Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

2013-01-01

The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak. PMID:23739677

The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus.

PubMed Central

Chan, H T; Anthony, C

1991-01-01

The quinoprotein methanol dehydrogenase (MDH) of Acetobacter methanolicus has an alpha 2 beta 2 structure. By contrast with other MDHs, the beta-subunit (approx. 8.5 kDa) does not contain the five lysine residues previously proposed to be involved in ionic interactions with the electron acceptor cytochrome cL. That electrostatic interactions are involved was confirmed by the demonstration that methanol:cytochrome cL oxidoreductase activity was inhibited by high ionic strength (I), the strength of interaction being inversely related to the square root of I. Specific modifiers of arginine residues on MDH inhibited this reaction but not the dye-linked MDH activity. Modification of lysine residues on MDH that altered its charge had no effect on the dye-linked activity but inhibited reaction with cytochrome cL. When the charge was retained on modification of lysine residues, little effect on either activity was observed. Cross-linking experiments confirmed that lysine residues on the alpha-subunit, but not the beta-subunit, are involved in the 'docking' process between the proteins. Images Fig. 4. PMID:1660263

Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

PubMed

Pinske, Constanze; Sargent, Frank

2016-10-01

During mixed-acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO 2 and H 2 . Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO 2 to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

PubMed

Vogel, O; Hoehn, B; Henning, U

1972-06-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

PubMed

Meng, Fantao; Xu, Yan

2010-04-01

An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

PubMed Central

Mansfeldt, Cresten B.; Rowe, Annette R.; Heavner, Gretchen L. W.; Zinder, Stephen H.

2014-01-01

A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 μM Cl−/h)- and fast (22.9 ± 9.6 μM Cl−/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID:25063656

Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase - Homology model, reactivity and isoenzymatic diversity.

PubMed

Rugor, Agnieszka; Wójcik-Augustyn, Anna; Niedzialkowska, Ewa; Mordalski, Stefan; Staroń, Jakub; Bojarski, Andrzej; Szaleniec, Maciej

2017-08-01

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O 2 -independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[ 2 H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

PubMed

Maj, Mary C; Raha, Sandeep; Myint, Tomoko; Robinson, Brian H

2004-01-01

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase.

PubMed

Weckbecker, Andrea; Hummel, Werner

2004-11-01

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD+ and NADPH. The pnta and pntb genes encoding for the alpha- and beta-subunits were cloned and co-expressed with NADP+-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12 h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

PubMed Central

Vogel, Otto; Hoehn, Barbara; Henning, Ulf

1972-01-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart.

PubMed Central

Nichols, B J; Rigoulet, M; Denton, R M

1994-01-01

The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available. PMID:7980405

Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

PubMed

Xekouki, Paraskevi; Stratakis, Constantine A

2012-12-01

Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney-Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD's loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects.

Mitochondrial Bioenergetics and Dysfunction in Failing Heart.

PubMed

Sheeran, Freya L; Pepe, Salvatore

2017-01-01

Energy insufficiency has been recognized as a key feature of systolic heart failure. Although mitochondria have long been known to sustain myocardial work energy supply, the capacity to therapeutically target mitochondrial bioenergetics dysfunction is hampered by a complex interplay of multiple perturbations that progressively compound causing myocardial failure and collapse. Compared to non-failing human donor hearts, activity rates of complexes I and IV, nicotinamide nucleotide transhydrogenase (NADPH-transhydrogenase, Nnt) and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase are markedly decreased in end-stage heart failure. Diminished REDOX capacity with lower total glutathione and coenzyme Q 10 levels are also a feature of chronic left ventricular failure. Decreased enzyme activities in part relate to abundant and highly specific oxidative, nitrosylative, and hyperacetylation modifications. In this brief review we highlight that energy deficiency in end-stage failing human left ventricle predominantly involves concomitantly impaired activities of key electron transport chain and Krebs cycle enzymes rather than altered expression of respective genes or proteins. Augmented oxidative modification of these enzyme subunit structures, and the formation of highly reactive secondary metabolites, implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, which contribute further to progressive decreases in bioenergetic capacity and contractile function in human heart failure.

Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

PubMed Central

Xekouki, Paraskevi; Stratakis, Constantine A

2013-01-01

Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney–Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD’s loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects. PMID:22889736

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

PubMed

Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

2004-01-01

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

PubMed

Gráczer, Eva; Varga, Andrea; Melnik, Bogdan; Semisotnov, Gennady; Závodszky, Péter; Vas, Mária

2009-02-10

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

PubMed

Pinske, Constanze

2018-01-01

Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H 2 and CO 2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H 2 -production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H 2 -production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA , but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H 2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Lipoic Acid Metabolism of Plasmodium - A Suitable Drug Target

PubMed Central

Storm, Janet; Müller, Sylke

2012-01-01

α-Lipoic acid (6,8-thioctic acid; LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. In recent years it was shown that biosynthesis and salvage of LA in Plasmodium are necessary for the parasites to complete their complex life cycle. LA salvage requires two lipoic acid protein ligases (LplA1 and LplA2). LplA1 is confined to the mitochondrion while LplA2 is located in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development suggesting a specific function for LplA2 that has yet to be elucidated. LA salvage is essential for the intra-erythrocytic and liver stage development of Plasmodium and thus offers great potential for future drug or vaccine development. LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP) : protein N-octanoyltransferase (LipB) and lipoate synthase (LipA), is exclusively found in the apicoplast of Plasmodium where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis (FAS II) pathway also present in the organelle. LA is the co-factor of the acetyltransferase subunit of the apicoplast located pyruvate dehydrogenase (PDH), which generates acetyl-CoA, feeding into FAS II. LA biosynthesis is not vital for intra-erythrocytic development of Plasmodium, but the deletion of several genes encoding components of FAS II or PDH was detrimental for liver stage development of the parasites indirectly suggesting that the same applies to LA biosynthesis. These data provide strong evidence that LA salvage and biosynthesis are vital for different stages of Plasmodium development and offer potential for drug and vaccine design against malaria. PMID:22607141

LDHk, an unusual oxygen-sensitive lactate dehydrogenase expressed in human cancer.

PubMed Central

Anderson, G R; Kovacik, W P

1981-01-01

An unusual isozyme of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27), LDHk, has been described in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme appears to contain one or more subunits encoded by the transforming gene of KiMSV and is readily distinguished from other isozymes of LDH. Specifically, it is more basic than other LDH isozymes, has an apparent subunit structure of (35,000)4(22,000)1, is essentially inactive if assayed under a normal atmosphere, and is strongly inhibited by GTP and various related compounds. We have examined human cancer and normal tissue controls for expression of an activity like LDHk. In 11 out of 16 human carcinomas, LDHk activity was increased 10- to 500-fold over the level seen in adjoining nontumor tissue. In contrast, other LDH isozymes were increased by only 2- to 5-fold. Images PMID:6942426

Myasthenia gravis: long-term prognostic value of thymus lactate dehydrogenase isoenzyme pattern of hyperplastic thymus and thymoma.

PubMed Central

Szathmáry, I; Selmeci, L; Pósch, E; Szobor, A; Molnár, J

1985-01-01

Lactate dehydrogenase (LDH) isoenzyme pattern and the percent of H-subunit content were determined in the thymus of 62 patients (55 with hyperplasia, 7 with tumours) after thymectomy. An increase in LDH1 relative activity indicates that in the thymus of patients with myasthenia gravis the ratio of mature differentiated thymocytes was higher than in the thymus of control subjects. LDH isoenzyme profiles of thymus tumours were similar to those described in other neoplasms, except that thymomas with apparent predominance of epithelial cells and with minimal lymphocytic reaction exhibited a marked elevation only in LDH2 relative activity, presumably associated with the specific (secretory) function of epithelial cells. The elevation of H-subunit content, a parameter characteristic of both thymic components (lymphoid and epithelial), correlated closely with a poor clinical condition in patients several years after surgery. PMID:4031927

Biochemical and molecular analysis of an X-linked case of Leigh syndrome associated with thiamin-responsive pyruvate dehydrogenase deficiency.

PubMed

Naito, E; Ito, M; Yokota, I; Saijo, T; Matsuda, J; Osaka, H; Kimura, S; Kuroda, Y

1997-08-01

We report molecular analysis of thiamin-responsive pyruvate dehydrogenase complex (PDHC) deficiency in a patient with an X-linked form of Leigh syndrome. PDHC activity in cultured lymphoblastoid cells of this patient and his asymptomatic mother were normal in the presence of a high thiamin pyrophosphate (TPP) concentration (0.4 mmol/L). However, in the presence of a low concentration (1 x 10(-4) mmol/L) of TPP, the activity was significantly decreased, indicating that PDHC deficiency in this patient was due to decreased affinity of PDHC for TPP. The patient's older brother also was diagnosed as PDHC deficiency with Leigh syndrome, suggesting that PDHC deficiency in these two brothers was not a de novo mutation. Sequencing of the X-linked PDHC E1 alpha subunit revealed a C-->G point mutation at nucleotide 787, resulting in a substitution of glycine for arginine 263. Restriction enzyme analysis of the E1 alpha gene revealed that the mother was a heterozygote, indicating that thiamin-responsive PDHC deficiency associated with Leigh syndrome due to this mutation is transmitted by X-linked inheritance.

Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Nedielkov, Ruslan; Wendelspiess, Severin; Vossler, Sara; Gerken, Uwe; Murai, Masatoshi; Miyoshi, Hideto; Möller, Heiko M.; Steuber, Julia

2011-01-01

Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA. PMID:21885438

An active Mitochondrial Complex II Present in Mature Seeds Contains an Embryo-Specific Iron-Sulfur Subunit Regulated by ABA and bZIP53 and Is Involved in Germination and Seedling Establishment.

PubMed

Restovic, Franko; Espinoza-Corral, Roberto; Gómez, Isabel; Vicente-Carbajosa, Jesús; Jordana, Xavier

2017-01-01

Complex II (succinate dehydrogenase) is an essential mitochondrial enzyme involved in both the tricarboxylic acid cycle and the respiratory chain. In Arabidopsis thaliana , its iron-sulfur subunit (SDH2) is encoded by three genes, one of them ( SDH2.3 ) being specifically expressed during seed maturation in the embryo. Here we show that seed SDH2.3 expression is regulated by abscisic acid (ABA) and we define the promoter region (-114 to +49) possessing all the cis -elements necessary and sufficient for high expression in seeds. This region includes between -114 and -32 three ABRE (ABA-responsive) elements and one RY-enhancer like element, and we demonstrate that these elements, although necessary, are not sufficient for seed expression, our results supporting a role for the region encoding the 5' untranslated region (+1 to +49). The SDH2.3 promoter is activated in leaf protoplasts by heterodimers between the basic leucine zipper transcription factors bZIP53 (group S1) and bZIP10 (group C) acting through the ABRE elements, and by the B3 domain transcription factor ABA insensitive 3 (ABI3). The in vivo role of bZIP53 is further supported by decreased SDH2.3 expression in a knockdown bzip53 mutant. By using the protein synthesis inhibitor cycloheximide and sdh2 mutants we have been able to conclusively show that complex II is already present in mature embryos before imbibition, and contains mainly SDH2.3 as iron-sulfur subunit. This complex plays a role during seed germination sensu-stricto since we have previously shown that seeds lacking SDH2.3 show retarded germination and now we demonstrate that low concentrations of thenoyltrifluoroacetone, a complex II inhibitor, also delay germination. Furthermore, complex II inhibitors completely block hypocotyl elongation in the dark and seedling establishment in the light, highlighting an essential role of complex II in the acquisition of photosynthetic competence and the transition from heterotrophy to autotrophy.

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES.

DTIC Science & Technology

The pig heart pyruvate and alpha - ketoglutarate dehydrogenase complex were isolated in highly purified state as multienzyme units with molecular...weights of approximately 9 million and 2.8 million, respectively. The aims were to resolve the pig heart pyruvate and alpha - ketoglutarate dehydrogenase...complexes was isolated from three sources; (1) pyruvate dehydrogenase complex, (2) alpha - ketoglutarate dehydrogenase, and (3) amber-color extract free

Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.

PubMed

Chowdhury, Nilanjan Pal; Mowafy, Amr M; Demmer, Julius K; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

2014-02-21

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans*

PubMed Central

Chowdhury, Nilanjan Pal; Mowafy, Amr M.; Demmer, Julius K.; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

2014-01-01

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD+ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH− is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH− by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD⨪, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH− that converts crotonyl-CoA to butyryl-CoA. PMID:24379410

Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

PubMed

Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

2014-07-01

Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simultaneous occurrence of the 11778 (ND4) and the 9438 (COX III) mtDNA mutations in Leber hereditary optic neuropathy: Molecular, biochemical, and clinical findings

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oostra, R.J.; Bleeker-Wagemakers, E.M.; Zwart, R.

1995-10-01

Three mtDNA point mutations at nucleotide position (np) 3460, at np 11778 and at np 14484, are thought to be of primary importance in the pathogenesis of Leber hereditary optic neuropathy (LHON), a maternally inherited disease characterized by subacute central vision loss. These mutations are present in genes coding for subunits of complex I (NADH dehydrogenase) of the respiratory chain, occur exclusively in LHON maternal pedigrees, and have never been reported to occur together. Johns and Neufeld postulated that an mtDNA mutation at np 9438, in the gene coding for one of the subunits (COX III) of complex IV (cytochromemore » c oxidase), was also of primary importance. Johns and Neufeld (1993) found this mutation, which changed a conserved glycine to a serine, in 5 unrelated LHON probands who did not carry one of the presently known primary mutations, but they did not find it in 400 controls. However, the role of this sequence variant has been questioned in the Journal when it has been found to occur in apparently healthy African and Cuban individuals. Subsequently, Johns et al. described this mutation in two Cuban individuals presenting with optic and peripheral neuropathy. 22 refs., 1 fig., 1 tab.« less

Changes in Whole-Body Oxygen Consumption and Skeletal Muscle Mitochondria During Linezolid-Induced Lactic Acidosis.

PubMed

Protti, Alessandro; Ronchi, Dario; Bassi, Gabriele; Fortunato, Francesco; Bordoni, Andreina; Rizzuti, Tommaso; Fumagalli, Roberto

2016-07-01

To better clarify the pathogenesis of linezolid-induced lactic acidosis. Case report. ICU. A 64-year-old man who died with linezolid-induced lactic acidosis. Skeletal muscle was sampled at autopsy to study mitochondrial function. Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA-encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Molecular, biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase.

PubMed

Kloosterman, Harm; Vrijbloed, Jan W; Dijkhuizen, Lubbert

2002-09-20

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism

PubMed Central

Gu, F; Chauhan, V; Kaur, K; Brown, W T; LaFauci, G; Wegiel, J; Chauhan, A

2013-01-01

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence suggests that mitochondrial dysfunction and oxidative stress may contribute to the etiology of autism. This is the first study to compare the activities of mitochondrial electron transport chain (ETC) complexes (I–V) and pyruvate dehydrogenase (PDH), as well as mitochondrial DNA (mtDNA) copy number in the frontal cortex tissues from autistic and age-matched control subjects. The activities of complexes I, V and PDH were most affected in autism (n=14) being significantly reduced by 31%, 36% and 35%, respectively. When 99% confidence interval (CI) of control group was taken as a reference range, impaired activities of complexes I, III and V were observed in 43%, 29% and 43% of autistic subjects, respectively. Reduced activities of all five ETC complexes were observed in 14% of autistic cases, and the activities of multiple complexes were decreased in 29% of autistic subjects. These results suggest that defects in complexes I and III (sites of mitochondrial free radical generation) and complex V (adenosine triphosphate synthase) are more prevalent in autism. PDH activity was also reduced in 57% of autistic subjects. The ratios of mtDNA of three mitochondrial genes ND1, ND4 and Cyt B (that encode for subunits of complexes I and III) to nuclear DNA were significantly increased in autism, suggesting a higher mtDNA copy number in autism. Compared with the 95% CI of the control group, 44% of autistic children showed higher copy numbers of all three mitochondrial genes examined. Furthermore, ND4 and Cyt B deletions were observed in 44% and 33% of autistic children, respectively. This study indicates that autism is associated with mitochondrial dysfunction in the brain. PMID:24002085

Mechanism of neem limonoids-induced cell death in cancer: role of oxidative phosphorylation

PubMed Central

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph; O’Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. PMID:26627937

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

PubMed

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Enhanced Iron Uptake of Saccharomyces cerevisiae by Heterologous Expression of a Tadpole Ferritin Gene

PubMed Central

Shin, Young-Mi; Kwon, Tae-Ho; Kim, Kyung-Suk; Chae, Keon-Sang; Kim, Dae-Hyuk; Kim, Jae-Ho; Yang, Moon-Sik

2001-01-01

We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement. PMID:11229922

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

NASA Astrophysics Data System (ADS)

Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

2014-11-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

A novel deficiency of mitochondrial ATPase of nuclear origin.

PubMed

Houstek, J; Klement, P; Floryk, D; Antonická, H; Hermanská, J; Kalous, M; Hansíková, H; Hout'ková, H; Chowdhury, S K; Rosipal, T; Kmoch, S; Stratilová, L; Zeman, J

1999-10-01

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

PubMed Central

Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

1975-01-01

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

PubMed

Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B; Barger, Philip M; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K

2016-01-01

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart. Collectively, these observations suggest that Med1 plays a critical role in the maintenance of heart function impacting on multiple metabolic, compensatory and reparative pathways with a likely therapeutic potential in the management of heart failure.

Human METTL20 Methylates Lysine Residues Adjacent to the Recognition Loop of the Electron Transfer Flavoprotein in Mitochondria*

PubMed Central

Rhein, Virginie F.; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

2014-01-01

In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. PMID:25023281

Human METTL20 methylates lysine residues adjacent to the recognition loop of the electron transfer flavoprotein in mitochondria.

PubMed

Rhein, Virginie F; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M; Walker, John E

2014-08-29

In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

White shrimp Litopenaeus vannamei recombinant lactate dehydrogenase: Biochemical and kinetic characterization.

PubMed

Fregoso-Peñuñuri, Ambar A; Valenzuela-Soto, Elisa M; Figueroa-Soto, Ciria G; Peregrino-Uriarte, Alma B; Ochoa-Valdez, Manuel; Leyva-Carrillo, Lilia; Yepiz-Plascencia, Gloria

2017-09-01

Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pK a of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants K m for NADH was 23.4 ± 1.8 μM, and for pyruvate was 203 ± 25 μM, while V max was 7.45 μmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

PubMed

Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

2017-01-15

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P) + -dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD + Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD + /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase. Copyright © 2016 American Society for Microbiology.

Identification of a novel mitochondrial complex I assembly factor ACDH-12 in Caenorhabditis elegans.

PubMed

Chuaijit, Sirithip; Boonyatistan, Worawit; Boonchuay, Pichsinee; Metheetrairut, Chanatip; Suthammarak, Wichit

2018-03-11

Assembly of complex I of the mitochondrial respiratory chain (MRC) requires not only structural subunits for electron transport, but also assembly factors. In the nematode Caenorhabditis elegans, NUAF-1 and NUAF-3 are the only two assembly factors that have been characterized. In this study, we identify ACDH-12 as an assembly factor of the respiratory complex I. We demonstrate for the first time that a deficiency of ACDH-12 affects the formation and function of complex I. RNAi knockdown of acdh-12 also shortens lifespan and decreases fecundity. Although ACDH-12 has long been recognized as a very long-chain acyl-CoA dehydrogenase (VLCAD), the knockdown nematodes did not exhibit any change in body fat content. We suggested that in Caenorhabditis elegans, ACDH-12 is required for the assembly of the respiratory complex I, but may not be crucial to fatty acid oxidation. Interestingly, sequence analysis shows high homology between ACDH-12 and the human ACAD9, a protein that has initially been identified as a VLCAD, but later found to also be involved in the assembly of complex I in human. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Transcontinental Phylogeography of the Daphnia pulex Species Complex

PubMed Central

Costanzo, Katie S.; Taylor, Derek J.

2012-01-01

Daphnia pulex is quickly becoming an attractive model species in the field of ecological genomics due to the recent release of its complete genome sequence, a wide variety of new genomic resources, and a rich history of ecological data. Sequences of the mitochondrial NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit 1 genes were used to assess the global phylogeography of this species, and to further elucidate its phylogenetic relationship to other members of the Daphnia pulex species complex. Using both newly acquired and previously published data, we analyzed 398 individuals from collections spanning five continents. Eleven strongly supported lineages were found within the D. pulex complex, and one lineage in particular, panarctic D. pulex, has very little phylogeographical structure and a near worldwide distribution. Mismatch distribution, haplotype network, and population genetic analyses are compatible with a North American origin for this lineage and subsequent spatial expansion in the Late Pleistocene. In addition, our analyses suggest that dispersal between North and South America of this and other species in the D. pulex complex has occurred multiple times, and is predominantly from north to south. Our results provide additional support for the evolutionary relationships of the eleven main mitochondrial lineages of the D. pulex complex. We found that the well-studied panarctic D. pulex is present on every continent except Australia and Antarctica. Despite being geographically very widespread, there is a lack of strong regionalism in the mitochondrial genomes of panarctic D. pulex – a pattern that differs from that of most studied cladocerans. Moreover, our analyses suggest recent expansion of the panarctic D. pulex lineage, with some continents sharing haplotypes. The hypothesis that hybrid asexuality has contributed to the recent and unusual geographic success of the panarctic D. pulex lineage warrants further study. PMID:23056371

Transcontinental phylogeography of the Daphnia pulex species complex.

PubMed

Crease, Teresa J; Omilian, Angela R; Costanzo, Katie S; Taylor, Derek J

2012-01-01

Daphnia pulex is quickly becoming an attractive model species in the field of ecological genomics due to the recent release of its complete genome sequence, a wide variety of new genomic resources, and a rich history of ecological data. Sequences of the mitochondrial NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit 1 genes were used to assess the global phylogeography of this species, and to further elucidate its phylogenetic relationship to other members of the Daphnia pulex species complex. Using both newly acquired and previously published data, we analyzed 398 individuals from collections spanning five continents. Eleven strongly supported lineages were found within the D. pulex complex, and one lineage in particular, panarctic D. pulex, has very little phylogeographical structure and a near worldwide distribution. Mismatch distribution, haplotype network, and population genetic analyses are compatible with a North American origin for this lineage and subsequent spatial expansion in the Late Pleistocene. In addition, our analyses suggest that dispersal between North and South America of this and other species in the D. pulex complex has occurred multiple times, and is predominantly from north to south. Our results provide additional support for the evolutionary relationships of the eleven main mitochondrial lineages of the D. pulex complex. We found that the well-studied panarctic D. pulex is present on every continent except Australia and Antarctica. Despite being geographically very widespread, there is a lack of strong regionalism in the mitochondrial genomes of panarctic D. pulex--a pattern that differs from that of most studied cladocerans. Moreover, our analyses suggest recent expansion of the panarctic D. pulex lineage, with some continents sharing haplotypes. The hypothesis that hybrid asexuality has contributed to the recent and unusual geographic success of the panarctic D. pulex lineage warrants further study.

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein.

PubMed

Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

2008-01-29

The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tasayco, M.L.; Prestwich, G.D.

1990-02-25

Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor ofmore » this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.« less

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2014-01-01

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

Sequence variability in three mitochondrial genes among four roundworm species from wild animals in China.

PubMed

Chang, Qiao-Cheng; Gao, Jun-Feng; Sheng, Zhong-Hua; Lou, Yan; Zheng, Xu; Wang, Chun-Ren

2015-02-01

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely portions of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and NADH dehydrogenase subunit 4 (pnad4), for Toxocara canis. Baylisacaris transfuga. Ascaris suum and Parascaris equorum from Canis lupus. Ursus thibetanus. Sus scrofa and Equus burchelli in China were examined. The lengths of the sequences of pcox1, pnad1 and pnad4 were 711 bp, 648 bp and 666 bp, respectively. No intra-species differences were detected in pcox1 for the four examined ascarid species, in pnad1 for T. canis. A. suum and P. equorum, and in pnad4 for B. transfuga and P. equorum. Sequence differences in pnad4 for six roundworm samples of T. canis and P. equorum were 0-0.1% and 0-0.3%, respectively, and were 0-0.3% in pnad1 for six roundworm samples isolate of B. transfuga. The inter-specific sequence differences among four species were 8.7-12.4% for pcox1, 13.9-17.7% for pnad1, and 14.0-25.7% for pnad4. Phylogenetic analyses suggested that the three mtDNA fragments could be used to identify ascarid species in families Ascaridiae and Toxocaridae.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

PubMed

Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

2016-01-01

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. © 2016 American Society of Plant Biologists. All Rights Reserved.

Succinate Dehydrogenase (SDH) D Subunit (SDHD) Inactivation in a Growth-Hormone-Producing Pituitary Tumor: A New Association for SDH?

PubMed Central

Xekouki, Paraskevi; Pacak, Karel; Almeida, Madson; Wassif, Christopher A.; Rustin, Pierre; Nesterova, Maria; de la Luz Sierra, Maria; Matro, Joey; Ball, Evan; Azevedo, Monalisa; Horvath, Anelia; Lyssikatos, Charalampos; Quezado, Martha; Patronas, Nicholas; Ferrando, Barbara; Pasini, Barbara; Lytras, Aristides; Tolis, George

2012-01-01

Background: Mutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD). Aim: Our aim was to examine the possible causative link between SDHD inactivation and somatotropinoma. Patients and Methods: A 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors. Conclusion: We describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHD's involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis. PMID:22170724

Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii

PubMed Central

Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

2014-01-01

The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. PMID:25210079

Demonstration of PDC-E1 subunits as major antigens in the complement-fixing fraction M4 and re-evaluation of PDC-E1-specific antibodies in PBC patients.

PubMed

Berg, Christoph P; Stein, Gerburg M; Klein, Reinhild; Pascu, Maria; Berg, Thomas; Kammer, Winfried; Priemer, Martin; Nordheim, Alfred; Schulze-Osthoff, Klaus; Gregor, Michael; Wesselborg, Sebastian; Berg, Peter A

2006-09-01

Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%. CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J L; Bal, Amanjit; Gill, Kiran Dip

2013-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. © 2013.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

PubMed

Chauhan, Indira Singh; Kaur, Jaspreet; Krishna, Shagun; Ghosh, Arpita; Singh, Prashant; Siddiqi, Mohammad Imran; Singh, Neeloo

2015-11-21

Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis.

PubMed

Cugno, Graziano; Parreira, José R; Ferlizza, Enea; Hernández-Castellano, Lorenzo E; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan; Campos, Alexandre M O; Almeida, André M

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

PubMed Central

Cugno, Graziano; Parreira, José R.; Ferlizza, Enea; Hernández-Castellano, Lorenzo E.; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss. PMID:27031334

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

PubMed

Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

2001-10-19

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding.

PubMed Central

Syed, S E; Hornby, D P; Brown, P E; Fitton, J E; Engel, P C

1994-01-01

Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources. PMID:8129708

Purification and characterization of the amine dehydrogenase from a facultative methylotroph.

PubMed

Coleman, J P; Perry, J J

1984-01-01

Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

Mitochondrial DNA sequences of 37 collar-spined echinostomes (Digenea: Echinostomatidae) in Thailand and Lao PDR reveals presence of two species: Echinostoma revolutum and E. miyagawai.

PubMed

Nagataki, Mitsuru; Tantrawatpan, Chairat; Agatsuma, Takeshi; Sugiura, Tetsuro; Duenngai, Kunyarat; Sithithaworn, Paiboon; Andrews, Ross H; Petney, Trevor N; Saijuntha, Weerachai

2015-10-01

The "37 collar-spined" or "revolutum" group of echinostomes is recognized as a species complex. The identification of members of this complex by morphological taxonomic characters is difficult and confusing, and hence, molecular analyses are a useful alternative method for molecular systematic studies. The current study examined the genetic diversity of those 37 collar-spined echinostomes which are recognized morphologically as Echinostoma revolutum in Thailand and Lao PDR using the cytochrome c oxidase subunit 1 (CO1) and the NADH dehydrogenase subunit 1 (ND1) sequences. On the basis of molecular investigations, at least two species of 37 collar-spined echinostomes exist in Southeast Asia, namely E. revolutum and Echinostoma miyagawai. The specimens examined in this study, coming from ducks in Thailand and Lao PDR, were compared to isolates from America, Europe and Australia for which DNA sequences are available in public databases. Haplotype analysis detected 6 and 26 haplotypes when comparing the CO1 sequences of E. revolutum and E. miyagawai, respectively, from different geographical isolates from Thailand and Lao PDR. The phylogenetic trees, ND1 haplotype network and genetic differentiation (ɸST) analyses showed that E. revolutum were genetically different on a continental scale, i.e. Eurasian and American lineages. Copyright © 2015 Elsevier B.V. All rights reserved.

Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

PubMed

Tamrakar, Pratistha; Ibrahim, Baher A; Gujar, Amit D; Briski, Karen P

2015-02-01

The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-β in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury. © 2014 Wiley Periodicals, Inc.

Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil.

PubMed

Pennacchio, Angela; Sannino, Vincenzo; Sorrentino, Giosuè; Rossi, Mosè; Raia, Carlo A; Esposito, Luciana

2013-05-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

PubMed

Mitchelson, K R

1996-01-01

The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

The absence of genes for cytochrome c oxidase and reductase subunits in maxicircle kinetoplast DNA of the respiration-deficient plant trypanosomatid Phytomonas serpens.

PubMed

Nawathean, P; Maslov, D A

2000-08-01

By completing the sequencing of the maxicircle conserved region in the kinetoplast DNA of Phytomonas serpens, we showed that the genes for subunits I and II (COI and COII) of cytochrome c oxidase in this organism were missing. We had previously shown that the genes for cytochrome c oxidase subunit III and apocytochrome b were also missing. These deletions did not affect the structure or expression of the remaining genes. Partial editing of the mRNA for NADH dehydrogenase subunit 8, previously found in strain IG from insects, was complete in two other strains isolated from plants. The appearance of a novel maxicircle gene for MURF2 block I gRNA, which substitutes for the gene missing due to the COII gene deletion, may illustrate a general mechanism for the origin of gRNAs.

Lipoic acid metabolism and mitochondrial redox regulation.

PubMed

Solmonson, Ashley D; DeBerardinis, Ralph J

2017-11-30

Lipoic acid is an essential cofactor for mitochondrial metabolism and is synthesized de novo using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a critical role in stabilizing and regulating these multi-enzyme complexes.  Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety.  Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity and coordination of fuel metabolism. Lipoic acid is an essential cofactor for mitochondrial metabolism and is synthesized de novo using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a critical role in stabilizing and regulating these multi-enzyme complexes.  Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety.  Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity and coordination of fuel metabolism. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

Structure-Derived Proton-Transfer Mechanism of Action Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2003-01-01

The derivative of vitamin B1 thiamin pyrophosphate (TPP) is a cofactor of pyruvate dehydrogenase (E1p) that is involved in decarboxylation of pyruvate followed by reductive acetylation of lipoic acid covalently bound to a lysine residue of dihydrolipoamide acetyltransferase. The structure of E1p recently determined in our laboratory revealed patterns of association of foul subunits and specifics of two TPP binding sites. The mechanism of action in part includes a conserved hydrogen bond between the N1' atom of the aminopyrimidine ring of the cofactor and the carboxylate group of Glu59 from the beta subunits, and a V-conformation of the cofactor that brings the N4' atom of the aminopyrimidine ring to the distance of the intramolecular hydrogen bond formed with the C2-atom of the thiazolium moiety. The carboxylate group of Glu59 is the local proton acceptor that enables proton translocation within the aminopyrimidine ring and stabilization of the rare N4' - iminopyrimidine tautomer. Based on the analysis of E1p structure, we postulate that the protein environment drives N4' - amino/N4' - imino dynamics resulting in a concerted shuttle-like movement of the subunits. We also propose that this movement of the subunits is strictly coordinated with the two enzymatic reactions carried out in E1p by each of the two cofactor sites. It is proposed that these reactions are in alternating phases such that when one active site is involved in decarboxylation, the other is involved in acetylation of lipoyl noiety.

Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high hetrogeneity

USDA-ARS?s Scientific Manuscript database

Fecal specimens from 120 lambs in Valencia (Spain) were analyzed for Giardia duodenalis by IFA and nested-PCR using the beta giardin, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes. The highest prevalence was obtained using the ssurRN...

Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-06-01

The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. © 2016 Wiley Periodicals, Inc.

Function and Regulation of the Formate Dehydrogenase Genes of the Methanogenic Archaeon Methanococcus maripaludis

PubMed Central

Wood, Gwendolyn E.; Haydock, Andrew K.; Leigh, John A.

2003-01-01

Methanococcus maripaludis is a mesophilic species of Archaea capable of producing methane from two substrates: hydrogen plus carbon dioxide and formate. To study the latter, we identified the formate dehydrogenase genes of M. maripaludis and found that the genome contains two gene clusters important for formate utilization. Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage. The first gene cluster encodes homologs of formate dehydrogenase α (FdhA) and β (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog. The second gene cluster encodes only FdhA and FdhB homologs. Mutants lacking either fdhA gene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate. Investigation of fdh gene expression revealed that transcription of both gene clusters is controlled by the presence of H2 and not by the presence of formate. PMID:12670979

Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients.

PubMed

Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho

2004-01-01

Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

NASA Technical Reports Server (NTRS)

2003-01-01

Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

Anaerobic Formate and Hydrogen Metabolism.

PubMed

Pinske, Constanze; Sawers, R Gary

2016-10-01

Numerous recent developments in the biochemistry, molecular biology, and physiology of formate and H2 metabolism and of the [NiFe]-hydrogenase (Hyd) cofactor biosynthetic machinery are highlighted. Formate export and import by the aquaporin-like pentameric formate channel FocA is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate. Formate is disproportionated by the reversible formate hydrogenlyase (FHL) complex, which has been isolated, allowing biochemical dissection of evolutionary parallels with complex I of the respiratory chain. A recently identified sulfido-ligand attached to Mo in the active site of formate dehydrogenases led to the proposal of a modified catalytic mechanism. Structural analysis of the homologous, H2-oxidizing Hyd-1 and Hyd-5 identified a novel proximal [4Fe-3S] cluster in the small subunit involved in conferring oxygen tolerance to the enzymes. Synthesis of Salmonella Typhimurium Hyd-5 occurs aerobically, which is novel for an enterobacterial Hyd. The O2-sensitive Hyd-2 enzyme has been shown to be reversible: it presumably acts as a conformational proton pump in the H2-oxidizing mode and is capable of coupling reverse electron transport to drive H2 release. The structural characterization of all the Hyp maturation proteins has given new impulse to studies on the biosynthesis of the Fe(CN)2CO moiety of the [NiFe] cofactor. It is synthesized on a Hyp-scaffold complex, mainly comprising HypC and HypD, before insertion into the apo-large subunit. Finally, clear evidence now exists indicating that Escherichia coli can mature Hyd enzymes differentially, depending on metal ion availability and the prevailing metabolic state. Notably, Hyd-3 of the FHL complex takes precedence over the H2-oxidizing enzymes.

Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

PubMed

Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

2015-06-01

Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

PubMed

Minchenko, O H; Riabovol, O O; Tsymbal, D O; Minchenko, D O; Ratushna, O O

2016-01-01

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

A new missense mutation in the BCKDHB gene causes the classic form of maple syrup urine disease (MSUD).

PubMed

Miryounesi, Mohammad; Ghafouri-Fard, Soudeh; Goodarzi, Hamedreza; Fardaei, Majid

2015-05-01

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disease caused by mutations in the BCKDHA, BCKDHB, DBT and DLD genes, which encode the E1α, E1β, E2 and E3 subunits of the branched chain α ketoacid dehydrogenase (BCKD) complex, respectively. This complex is involved in the metabolism of branched-chain amino acids. In this study, we analyzed the DNA sequences of BCKDHA and BCKDHB genes in an infant who suffered from MSUD and died at the age of 6 months. We found a new missense mutation in exon 5 of BCKDHB gene (c.508C>T). The heterozygosity of the parents for the mentioned nucleotide change was confirmed by direct sequence analysis of the corresponding segment. Another missense mutation has been found in the same codon previously and shown by in silico analyses to be deleterious. This report provides further evidence that this amino acid change can cause classic MSUD.

Effects of clofibric acid on the activity and activity state of the hepatic branched-chain 2-oxo acid dehydrogenase complex.

PubMed Central

Zhao, Y; Jaskiewicz, J; Harris, R A

1992-01-01

Feeding clofibric acid to rats caused little or no change in total activity of the liver branched-chain 2-oxo acid dehydrogenase complex (BCODC). No change in mass of liver BCODC was detected by immunoblot analysis in response to dietary clofibric acid. No changes in abundance of mRNAs for the BCODC E1 alpha, E1 beta and E2 subunits were detected by Northern-blot analysis. Likewise, dietary clofibric acid had no effect on the activity state of liver BCODC (percentage of enzyme in the dephosphorylated, active, form) of rats fed on a chow diet. However, dietary clofibric acid greatly increased the activity state of liver BCODC of rats fed on a diet deficient in protein. No stable change in liver BCODC kinase activity was found in response to clofibric acid in either chow-fed or low-protein-fed rats. Clofibric acid had a biphasic effect on flux through BCODC in hepatocytes prepared from low-protein-fed rats. Stimulation of BCODC flux at low concentrations was due to clofibric acid inhibition of BCODC kinase, which in turn allowed activation of BCODC by BCODC phosphatase. Inhibition of BCODC flux at high concentrations was due to direct inhibition of BCODC by clofibric acid. The results suggest that the effects of clofibric acid in vivo on branched-chain amino acid metabolism can be explained by the inhibitory effects of this drug on BCODC kinase. Images Fig. 2. Fig. 3. PMID:1637295

Response of a rice paddy soil methanogen to syntrophic growth as revealed by transcriptional analyses.

PubMed

Liu, Pengfei; Yang, Yanxiang; Lü, Zhe; Lu, Yahai

2014-08-01

Members of Methanocellales are widespread in paddy field soils and play the key role in methane production. These methanogens feature largely in these organisms’ adaptation to low H2 and syntrophic growth with anaerobic fatty acid oxidizers. The adaptive mechanisms, however, remain unknown. In the present study, we determined the transcripts of 21 genes involved in the key steps of methanogenesis and acetate assimilation of Methanocella conradii HZ254, a strain recently isolated from paddy field soil. M. conradii was grown in monoculture and syntrophically with Pelotomaculum thermopropionicum (a propionate syntroph) or Syntrophothermus lipocalidus (a butyrate syntroph). Comparison of the relative transcript abundances showed that three hydrogenase-encoding genes and all methanogenesis-related genes tested were upregulated in cocultures relative to monoculture. The genes encoding formylmethanofuran dehydrogenase (Fwd), heterodisulfide reductase (Hdr), and the membrane-bound energy-converting hydrogenase (Ech) were the most upregulated among the evaluated genes. The expression of the formate dehydrogenase (Fdh)-encoding gene also was significantly upregulated. In contrast, an acetate assimilation gene was downregulated in cocultures. The genes coding for Fwd, Hdr, and the D subunit of F420-nonreducing hydrogenase (Mvh) form a large predicted transcription unit; therefore, the Mvh/Hdr/Fwd complex, capable of mediating the electron bifurcation and connecting the first and last steps of methanogenesis, was predicted to be formed in M. conradii. We propose that Methanocella methanogens cope with low H2 and syntrophic growth by (i) stabilizing the Mvh/Hdr/Fwd complex and (ii) activating formatedependent methanogenesis.

Insight into Energy Conservation via Alternative Carbon Monoxide Metabolism in Carboxydothermus pertinax Revealed by Comparative Genome Analysis.

PubMed

Fukuyama, Yuto; Omae, Kimiho; Yoneda, Yasuko; Yoshida, Takashi; Sako, Yoshihiko

2018-05-04

Carboxydothermus species are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via CO metabolism. In this study, we used comparative genome analysis of the genus Carboxydothermus to show variations in the CO dehydrogenase/energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H 2 production (hydrogenogenic CO metabolism). Indeed, ability or inability to produce H 2 with CO oxidation is explained by the presence or absence of this gene cluster in C. hydrogenoformans , C. islandicus , and C. ferrireducens Interestingly, despite its hydrogenogenic CO metabolism, C. pertinax lacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator encoding genes in this gene cluster probably due to inversion. Transcriptional analysis in C. pertinax showed that the Ni-CO dehydrogenase gene ( cooS-II ) and distantly encoded energy-converting hydrogenase related genes were remarkably upregulated under 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor under 100% CO, C. pertinax maximum cell density and maximum specific growth rate were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H 2 produced was only 63% of the consumed CO, less than expected according to hydrogenogenic CO oxidation: CO + H 2 O → CO 2 + H 2 Accordingly, C. pertinax would couple CO oxidation by Ni-CO dehydrogenase-II with simultaneous reduction of not only H 2 O but thiosulfate when grown under 100% CO. IMPORTANCE Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche with scavenging potentially toxic CO and producing H 2 as available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase/energy-converting hydrogenase gene cluster. This feature is thought to be as common to these organisms. However, hydrogenogenic carboxydotroph, Carboxydothermus pertinax lacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genus Carboxydothermus , transcriptional analysis, and cultivation study under 100% CO to prove their hydrogenogenic CO metabolism. Results revealed that C. pertinax could couple Ni-CO dehydrogenase-II alternatively to the distal energy-converting hydrogenase. Furthermore, C. pertinax represents an example of the functioning of Ni-CO dehydrogenase which does not always correspond with its genomic context owing to the versatility of CO metabolism and the low redox potential of CO. Copyright © 2018 American Society for Microbiology.

Glutathionylation of α-ketoglutarate dehydrogenase: the chemical nature and relative susceptibility of the cofactor lipoic acid to modification.

PubMed

McLain, Aaron L; Cormier, Peter J; Kinter, Michael; Szweda, Luke I

2013-08-01

α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione, resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly, producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione-lipoic acid adduct. Nevertheless, 1:1 lipoic acid-glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme's native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition, providing further evidence that specific lipoic acid residues within the E2 complex are susceptible to glutathionylation. These findings offer chemical information necessary for the identification of mechanisms and physiological implications of KGDH glutathionylation. Copyright © 2013 Elsevier Inc. All rights reserved.

Glutathionylation of α-ketoglutarate dehydrogenase: The chemical nature and relative susceptibility of the cofactor lipoic acid to modification

PubMed Central

McLain, Aaron L.; Cormier, Peter J.; Kinter, Michael; Szweda, Luke I.

2013-01-01

α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione, resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly, producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione–lipoic acid adduct. Nevertheless, 1:1 lipoic acid–glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme’s native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition, providing further evidence that specific lipoic acid residues within the E2 complex are susceptible to glutathionylation. These findings offer chemical information necessary for the identification of mechanisms and physiological implications of KGDH glutathionylation. PMID:23567190

Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus.

PubMed

Laczkovich, Irina; Teoh, Wei Ping; Flury, Sarah; Grayczyk, James P; Zorzoli, Azul; Alonzo, Francis

2018-04-16

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH-L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α-ketoacid dehydrogenase E2 subunits, while the function of GcvH-L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain-containing proteins; LplA1 targets H proteins and LplA2 targets α-ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH-L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation. © 2018 John Wiley & Sons Ltd.

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

NASA Astrophysics Data System (ADS)

Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

2003-06-01

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

Intraspecific Variation of Eysarcoris guttigerus (Hemiptera: Pentatomidae) in Japanese Southwest Population Based on Mitochondrial DNA

PubMed Central

Yamaji, Takuya; Ishikawa, Tadashi; Nomura, Masashi

2016-01-01

The white-spotted globular bug Eysarcoris guttigerus (Thunberg) (Hemiptera: Pentatomidae) is widely distributed in East Asia and the Pacific region. In Japan, the species is found in grassy or composite weeds in the western area of the main islands and Ryukyu Islands of Japan. One notable characteristic of the Eysarcoris genus is the two white spots on the scutellum. This is not the case with the Ishigaki Island population, however, which sports red spots instead of white, suggesting that intraspecific variation exists in the species. Therefore, we investigated intraspecific variation in E. guttigerus using mitochondrial NADH dehydrogenase subunit 2 (ND2), cytochrome oxidase subunit 1 (CO1), cytochrome b (Cytb), tRNA-Serine (tRNAser), NADH dehydrogenase subunit 1 (ND1), and 16S ribosomal RNA (16SrRNA) genes from 13 populations of Japan. The obtained maximum likelihood phylogenetic tree was divided into three groups—Group 1: Mainland, Group 2: Central Ryukyu Islands (Okinawa-Amamioshima Islands), and Group 3: South Ryukyu Islands (Ishigaki Island). The Ishigaki population was significantly separated from the other populations with consistent differences in spot color. The estimated period of divergence between the Ishigaki population and the other populations was consistent with the period of formation of the Kerama Gap in the Ryukyu arc. Thus, the process of formation of the Kerama Gap may have influenced the intraspecific variation of E. guttigerus. PMID:26798143

Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

In silico prediction of the pathogenic effect of a novel variant of BCKDHA leading to classical maple syrup urine disease identified using clinical exome sequencing.

PubMed

Fernández-Lainez, Cynthia; Aláez-Verson, Carmen; Ibarra-González, Isabel; Enríquez-Flores, Sergio; Carrillo-Sanchez, Karol; Flores-Lagunes, Leonardo; Guillén-López, Sara; Belmont-Martínez, Leticia; Vela-Amieva, Marcela

2018-04-16

Maple syrup urine disease (MSUD) is a metabolic disorder caused by mutations in three of the branched-chain α-keto acid dehydrogenase complex (BCKDC) genes. Classical MSUD symptom can be observed immediately after birth and include ketoacidosis, irritability, lethargy, and coma, which can lead to death or irreversible neurodevelopmental delay in survivors. The molecular diagnosis of MSUD can be time-consuming and difficult to establish using conventional Sanger sequencing because it could be due to pathogenic variants of any of the BCKDC genes. Next-generation sequencing-based methodologies have revolutionized the molecular diagnosis of inborn errors in metabolism and offer a superior approach for genotyping these patients. Here, we report an MSUD case whose molecular diagnosis was performed by clinical exome sequencing (CES), and the possible structural pathogenic effect of a novel E1α subunit pathogenic variant was analyzed using in silico analysis of α and β subunit crystallographic structure. Molecular analysis revealed a new homozygous non-sense c.1267C>T or p.Gln423Ter variant of BCKDHA. The novel BCKDHA variant is considered pathogenic because it caused a premature stop codon that probably led to the loss of the last 22 amino acid residues of the E1α subunit C-terminal end. In silico analysis of this region showed that it is in contact with several residues of the E1β subunit mainly through polar contacts, hydrogen bonds, and hydrophobic interactions. CES strategy could benefit the patients and families by offering precise and prompt diagnosis and better genetic counseling. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

PubMed Central

Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

2008-01-01

Background The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. Results In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. Conclusion These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging. PMID:18230186

Bilateral pheochromocytoma associated with paraganglioma and papillary thyroid carcinoma: report of an unusual case.

PubMed

Yang, Jeong Hoon; Bae, Sung Jin; Park, Sanghui; Park, Hyun-Kyung; Jung, Hye Seung; Chung, Jae Hoon; Min, Yong-Ki; Lee, Myung-Shik; Kim, Kwang-Won; Lee, Moon-Kyu

2007-04-01

A 42-year old woman presented with headache, palpitation and facial flushing. Ultrasonograms and computed tomograms revealed tumors in both of the adrenal glands, anterior aspect of the inferior vena cava, and the right lobe of the thyroid gland. Fine needle aspiration biopsy of the thyroid nodule revealed papillary thyroid carcinoma. Serum calcitonin, CEA, intact PTH and calcium levels were within normal limits. Markedly elevated levels of urinary normetanephrine and vanillylmandelic acid, and the result of 131I-metaiodobenzylguanidine (131I-MIBG) scintigraphy indicated that both adrenal masses were pheochromocytoma. Bilateral adrenalectomy, paracaval mass removal and total thyroidectomy together with central lymph node dissection were performed. The final pathological diagnosis was bilateral adrenal pheochromocytoma, paraganglioma, papillary thyroid carcinoma and either parathyroid adenoma or hyperplasia. Analysis of the RET proto-oncogene mutation, von Hippel Lindau mutation, succinate dehydrogenase subunit B mutation, and succinate dehydrogenase subunit D mutation yielded negative results. The relationship of these lesions could not be determined. This is the first report of a combination of bilateral pheochromocytoma, abdominal paraganglioma, papillary thyroid carcinoma and either parathyroid adenoma or hyperplasia without hyperparathyroidism.

Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs.

PubMed

Holmes, Roger S; Goldberg, Erwin

2009-10-01

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals.

Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs

PubMed Central

Holmes, Roger S; Goldberg, Erwin

2009-01-01

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals. PMID:19679512

Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

PubMed

Lewis, Brian A

2010-01-15

The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

PubMed Central

Kaufmann, Franz; Lovley, Derek R.

2001-01-01

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content. PMID:11443080

Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plapp, Bryce V.; Ramaswamy, S.; Iowa)

2013-01-16

Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 andmore » Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.« less

Insight to the Interaction of the Dihydrolipoamide Acetyltransferase (E2) Core with the Peripheral Components in the Escherichia coli Pyruvate Dehydrogenase Complex via Multifaceted Structural Approaches*

PubMed Central

Chandrasekhar, Krishnamoorthy; Wang, Junjie; Arjunan, Palaniappa; Sax, Martin; Park, Yun-Hee; Nemeria, Natalia S.; Kumaran, Sowmini; Song, Jaeyoung; Jordan, Frank; Furey, William

2013-01-01

Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 Å resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex. PMID:23580650

Insight to the interaction of the dihydrolipoamide acetyltransferase (E2) core with the peripheral components in the Escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches.

PubMed

Chandrasekhar, Krishnamoorthy; Wang, Junjie; Arjunan, Palaniappa; Sax, Martin; Park, Yun-Hee; Nemeria, Natalia S; Kumaran, Sowmini; Song, Jaeyoung; Jordan, Frank; Furey, William

2013-05-24

Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 Å resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.

Palladium alpha-lipoic acid complex formulation enhances activities of Krebs cycle dehydrogenases and respiratory complexes I-IV in the heart of aged rats.

PubMed

Sudheesh, N P; Ajith, T A; Janardhanan, K K; Krishnan, C V

2009-08-01

Age-related decline in the capacity to withstand stress, such as ischemia and reperfusion, results in congestive heart failure. Though the mechanisms underlying cardiac decay are not clear, age dependent somatic damages to mitochondrial DNA (mtDNA), loss of mitochondrial function, and a resultant increase in oxidative stress in heart muscle cells may be responsible for the increased risk for cardiovascular diseases. The effect of a safe nutritional supplement, POLY-MVA, containing the active ingredient palladium alpha-lipoic acid complex, was evaluated on the activities of the Krebs cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV in heart mitochondria of aged male albino rats of Wistar strain. Administration of 0.05 ml/kg of POLY-MVA (which is equivalent to 0.38 mg complexed alpha-lipoic acid/kg, p.o), once daily for 30 days, was significantly (p<0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complexes. The unique electronic and redox properties of palladium alpha-lipoic acid complex appear to be a key to this physiological effectiveness. The results strongly suggest that this formulation might be effective to protect the aging associated risk of cardiovascular and neurodegenerative diseases.

Comparative genomics of Fructobacillus spp. and Leuconostoc spp. reveals niche-specific evolution of Fructobacillus spp.

DOE PAGES

Endo, Akihito; Tanizawa, Yasuhiro; Tanaka, Naoto; ...

2015-12-29

In this study, Fructobacillus spp. in fructose-rich niches belong to the family Leuconostocaceae. They were originally classified as Leuconostoc spp., but were later grouped into a novel genus, Fructobacillus , based on their phylogenetic position, morphology and specific biochemical characteristics. The unique characters, so called fructophilic characteristics, had not been reported in the group of lactic acid bacteria, suggesting unique evolution at the genome level. Here we studied four draft genome sequences of Fructobacillus spp. and compared their metabolic properties against those of Leuconostoc spp. As a result, Fructobacillus species possess significantly less protein coding sequences in their small genomes.more » The number of genes was significantly smaller in carbohydrate transport and metabolism. Several other metabolic pathways, including TCA cycle, ubiquinone and other terpenoid-quinone biosynthesis and phosphotransferase systems, were characterized as discriminative pathways between the two genera. The adhE gene for bifunctional acetaldehyde/alcohol dehydrogenase, and genes for subunits of the pyruvate dehydrogenase complex were absent in Fructobacillus spp. The two genera also show different levels of GC contents, which are mainly due to the different GC contents at the third codon position. In conclusion, the present genome characteristics in Fructobacillus spp. suggest reductive evolution that took place to adapt to specific niches.« less

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

PubMed Central

Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

2004-01-01

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

Comparative genomics of Fructobacillus spp. and Leuconostoc spp. reveals niche-specific evolution of Fructobacillus spp.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Endo, Akihito; Tanizawa, Yasuhiro; Tanaka, Naoto

In this study, Fructobacillus spp. in fructose-rich niches belong to the family Leuconostocaceae. They were originally classified as Leuconostoc spp., but were later grouped into a novel genus, Fructobacillus , based on their phylogenetic position, morphology and specific biochemical characteristics. The unique characters, so called fructophilic characteristics, had not been reported in the group of lactic acid bacteria, suggesting unique evolution at the genome level. Here we studied four draft genome sequences of Fructobacillus spp. and compared their metabolic properties against those of Leuconostoc spp. As a result, Fructobacillus species possess significantly less protein coding sequences in their small genomes.more » The number of genes was significantly smaller in carbohydrate transport and metabolism. Several other metabolic pathways, including TCA cycle, ubiquinone and other terpenoid-quinone biosynthesis and phosphotransferase systems, were characterized as discriminative pathways between the two genera. The adhE gene for bifunctional acetaldehyde/alcohol dehydrogenase, and genes for subunits of the pyruvate dehydrogenase complex were absent in Fructobacillus spp. The two genera also show different levels of GC contents, which are mainly due to the different GC contents at the third codon position. In conclusion, the present genome characteristics in Fructobacillus spp. suggest reductive evolution that took place to adapt to specific niches.« less

Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

PubMed

Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

2017-07-14

The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Overexpression of hypoxia-inducible factor and metabolic pathways: possible targets of cancer.

PubMed

Singh, Davinder; Arora, Rohit; Kaur, Pardeep; Singh, Balbir; Mannan, Rahul; Arora, Saroj

2017-01-01

Cancer, the main cause of human deaths in the modern world is a group of diseases. Anticancer drug discovery is a challenge for scientists because of involvement of multiple survival pathways of cancer cells. An extensive study on the regulation of each step of these pathways may help find a potential cancer target. Up-regulated HIF-1 expression and altered metabolic pathways are two classical characteristics of cancer. Oxygen-dependent (through pVHL, PHDs, calcium-mediated) and independent (through growth factor signaling pathway, mdm2 pathway, HSP90) regulation of HIF-1α leads to angiogenesis, metastasis, and cell survival. The two subunits of HIF-1 regulates in the same fashion through different mechanisms. HIF-1α translation upregulates via mammalian target of rapamycin and mitogen-activated protein kinase signaling pathways, whereas HIF-1β through calmodulin kinase. Further, the stabilized interactions of these two subunits are important for proper functioning. Also, metabolic pathways crucial for the formation of building blocks (pentose phosphate pathway) and energy generation (glycolysis, TCA cycle and catabolism of glutamine) are altered in cancer cells to protect them from oxidative stress and to meet the reduced oxygen and nutrient supply. Up-regulated anaerobic metabolism occurs through enhanced expression of hexokinase, phosphofructokinase, triosephosphate isomerase, glucose 6-phosphate dehydrogenase and down-regulation of aerobic metabolism via pyruvate dehydrogenase kinase and lactate dehydrogenase which compensate energy requirements along with high glucose intake. Controlled expression of these two pathways through their common intermediate may serve as potent cancer target in future.

Branched-Chain Amino and Keto Acid Biochemistry and Cellular Biology in Central Nervous System Diseases

DTIC Science & Technology

2009-05-21

pyruvate dehydrogenase complex (PDC) and 2-oxo- glutarate dehydrogenase complex. These dehydrogenase complexes share the same basic structure, perform the...Science 312 (2006) 927-930. [20] J. Dancis, M. Levitz, R.G. Westall, Maple syrup urine disease: branched- chain keto- aciduria , Pediatrics 25 (1960...2127 2128 Dancis J, Levitz M, Westall RG. 1960. Maple syrup urine disease: branched-chain keto- aciduria . Pediatrics 25:72-9. Danner DJ, Lemmon

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.

PubMed

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-02-01

The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.

Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain of Arabidopsis Mitochondria[W][OA

PubMed Central

Araújo, Wagner L.; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Larson, Tony R.; Tohge, Takayuki; Krahnert, Ina; Witt, Sandra; Obata, Toshihiro; Schauer, Nicolas; Graham, Ian A.; Leaver, Christopher J.; Fernie, Alisdair R.

2010-01-01

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route. PMID:20501910

Identification of the 2-hydroxyglutarate and isovaleryl-CoA dehydrogenases as alternative electron donors linking lysine catabolism to the electron transport chain of Arabidopsis mitochondria.

PubMed

Araújo, Wagner L; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Larson, Tony R; Tohge, Takayuki; Krahnert, Ina; Witt, Sandra; Obata, Toshihiro; Schauer, Nicolas; Graham, Ian A; Leaver, Christopher J; Fernie, Alisdair R

2010-05-01

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

PubMed Central

2011-01-01

Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work. Conclusions In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction. PMID:21241518

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

PubMed Central

Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

2011-01-01

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes.

PubMed

Moody, Rebecca Reed; Lo, Miao-Chia; Meagher, Jennifer L; Lin, Chang-Ching; Stevers, Nicholas O; Tinsley, Samantha L; Jung, Inkyung; Matvekas, Aleksas; Stuckey, Jeanne A; Sun, Duxin

2018-02-09

The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strand, Deserah D.; Livingston, Aaron K.; Satoh-Cruz, Mio

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H 2O 2 . Surprisingly, hcef2 was mapped to a nonsense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codonmore » recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash induced thylakoid electric field suggest that these defect lead to accumulation of H 2O 2 in hcef2, which we have previously shown leads to activation of NDHrelated CEF. We observed similar increases in CEF and H 2O 2 accumulation in other translation defective mutants, suggesting that loss of coordination in plastid protein levels lead to imbalances in the photosynthetic energy balance that leads to increased CEF. These results, together with a large body of previous observations, support a general model in which processes that imbalances in chloroplast energetics result in the production of H 2O 2 , which activates CEF, either as a redox signal or by inducing deficits in ATP levels.« less

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

PubMed Central

Senior, Alan E.; Muharemagi, Alma; Wilke-Mounts, Susan

2008-01-01

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F1-subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector. PMID:17176112

Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex

PubMed Central

Wei, Kai; Zhang, Haiyan; Zhang, Yuewei; Xu, Jian; Jiang, Fei; Liu, Xu; Xu, Wei; Wu, Wenxue

2014-01-01

The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis. PMID:24520369

Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

PubMed

Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

2014-12-01

The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

PDPR Gene Expression Correlates with Exercise-Training Insulin Sensitivity Changes

PubMed Central

Barberio, Matthew D.; Huffman, Kim M.; Giri, Mamta; Hoffman, Eric P.; Kraus, William E.; Hubal, Monica J.

2016-01-01

Purpose Whole body insulin sensitivity (Si) typically improves following aerobic exercise training; however, individual responses can be highly variable. The purpose of this study was to use global gene expression to identify skeletal muscle genes that correlate with exercise-induced Si changes. Methods Longitudinal cohorts from the Studies of Targeted Risk Reduction Intervention through Defined Exercise (STRRIDE) were utilized as Discovery (Affymetrix) and Confirmation (Illumina) of vastus lateralis gene expression profiles. Discovery (n=39; 21 men) and Confirmation (n=42; 19 men) cohorts were matched for age (52 ± 8 vs. 51 ± 10 yr), BMI (30.4 ± 2.8 vs. 29.7 ± 2.8 kg*m-2), and VO2max (30.4 ± 2.8 vs. 29.7 ± 2.8 mL/kg/min). Si was determined via intravenous glucose tolerance test pre- and post-training. Pearson product-moment correlation coefficients determined relationships between a) baseline and b) training-induced changes in gene expression and %ΔSi after training. Results Expression of 2454 (Discovery) and 1778 genes (Confirmation) at baseline were significantly (P<0.05) correlated to %ΔSi; 112 genes overlapped. Pathway analyses identified Ca2+-signaling-related transcripts in this 112-gene list. Expression changes of 1384 (Discovery) and 1288 genes (Confirmation) following training were significantly (P<0.05) correlated to % ΔSi; 33 genes overlapped, representing contractile apparatus of skeletal and smooth muscle genes. Pyruvate dehydrogenase phosphatase regulatory subunit (PDPR) expression at baseline (p=0.01, r=0.41) and post-training (p=0.01, r=0.43) were both correlated with %ΔSi. Conclusion Exercise-induced adaptations in skeletal muscle Si are related to baseline levels of Ca+2-regulating transcripts, which may prime the muscle for adaptation. Relationships between %ΔSi and PDPR, a regulatory subunit of the pyruvate dehydrogenase complex, indicate that the Si response is strongly related to key steps in metabolic regulation. PMID:27846149

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

PubMed Central

Uchino, Yuichi; Kawakita, Tetsuya; Miyazawa, Masaki; Ishii, Takamasa; Onouchi, Hiromi; Yasuda, Kayo; Ogawa, Yoko; Shimmura, Shigeto; Ishii, Naoaki; Tsubota, Kazuo

2012-01-01

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease. PMID:23071526

Enhanced hydrogen production from formic acid by formate hydrogen lyase-overexpressing Escherichia coli strains.

PubMed

Yoshida, Akihito; Nishimura, Taku; Kawaguchi, Hideo; Inui, Masayuki; Yukawa, Hideaki

2005-11-01

Genetic recombination of Escherichia coli in conjunction with process manipulation was employed to elevate the efficiency of hydrogen production in the resultant strain SR13 2 orders of magnitude above that of conventional methods. The formate hydrogen lyase (FHL)-overexpressing strain SR13 was constructed by combining FHL repressor (hycA) inactivation with FHL activator (fhlA) overexpression. Transcription of large-subunit formate dehydrogenase, fdhF, and large-subunit hydrogenase, hycE, in strain SR13 increased 6.5- and 7.0-fold, respectively, compared to the wild-type strain. On its own, this genetic modification effectively resulted in a 2.8-fold increase in hydrogen productivity of SR13 compared to the wild-type strain. Further enhancement of productivity was attained by using a novel method involving the induction of the FHL complex with high-cell-density filling of a reactor under anaerobic conditions. Continuous hydrogen production was achieved by maintaining the reactor concentration of the substrate (free formic acid) under 25 mM. An initial productivity of 23.6 g hydrogen h(-1) liter(-1) (300 liters h(-1) liter(-1) at 37 degrees C) was achieved using strain SR13 at a cell density of 93 g (dry weight) cells/liter. The hydrogen productivity reported in this work has great potential for practical application.

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

PubMed Central

Baron, S F; Franklund, C V; Hylemon, P B

1991-01-01

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7 alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gt11, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7 alpha-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7 alpha-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5 alpha transformant harboring pBH51 had approximately 30-fold greater levels of 7 alpha-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7 alpha-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reached with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding. Images PMID:1856160

Specific roles for the Ccr4-Not complex subunits in expression of the genome

PubMed Central

Azzouz, Nowel; Panasenko, Olesya O.; Deluen, Cécile; Hsieh, Julien; Theiler, Grégory; Collart, Martine A.

2009-01-01

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit's function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome. PMID:19155328

Identification and transcription profiling of NDUFS8 in Aedes taeniorhynchus (Diptera:Culididae): developmental regulation and environmental response

USDA-ARS?s Scientific Manuscript database

The cDNA of a NADH dehydrogenase -ubiquinone Fe-S protein 8 subunit (NDUFS8) gene from Aedes (Ochlerotatus) taeniorhynchus Wiedemann has been cloned and sequenced. The full-length mRNA sequence (824 bp) of AetNDUFS8 encodes an open reading region of 651 bp (i.e., 217 amino acids). To detect whether ...

Failure of MIBG scan to detect metastases in SDHB-mutated pediatric metastatic pheochromocytoma.

PubMed

Sait, Sameer; Pandit-Taskar, Neeta; Modak, Shakeel

2017-11-01

123 I-meta-iodo benzyl guanidine (MIBG) scans are considered the gold standard imaging in neuroblastoma; however, flouro deoxy glucose positron emission tomography (FDG-PET) scans have increased sensitivity in adults with pheochromocytoma/paraganglioma. We describe a pediatric patient initially considered to have localized neuroblastoma based on anatomical imaging and 123 I-MIBG scan, but subsequent investigations revealed germline succinate dehydrogenase complex iron sulfur subunit B (SDHB) mutation-associated pheochromocytoma with multiple FDG-avid skeletal metastases. We then compared 123 I-MIBG and FDG-PET scans in children with metastatic pheochromocytoma/paraganglioma. FDG-PET was superior to 123 I-MIBG scan for the detection of skeletal metastases (median number of skeletal lesions detected 10 [range 1-30] vs. 2 [range 1-26], respectively; P = 0.005 by t-test). FDG-PET should be considered the functional scan of choice in children with pheochromocytoma/paraganglioma. © 2017 Wiley Periodicals, Inc.

A rapid screening with direct sequencing from blood samples for the diagnosis of Leigh syndrome.

PubMed

Shimbo, Hiroko; Takagi, Mariko; Okuda, Mitsuko; Tsuyusaki, Yu; Takano, Kyoko; Iai, Mizue; Yamashita, Sumimasa; Murayama, Kei; Ohtake, Akira; Goto, Yu-Ichi; Aida, Noriko; Osaka, Hitoshi

2014-01-01

Large numbers of genes are responsible for Leigh syndrome (LS), making genetic confirmation of LS difficult. We screened our patients with LS using a limited set of 21 primers encompassing the frequently reported gene for the respiratory chain complexes I (ND1-ND6, and ND4L), IV(SURF1), and V(ATP6) and the pyruvate dehydrogenase E1α-subunit. Of 18 LS patients, we identified mutations in 11 patients, including 7 in mDNA (two with ATP6), 4 in nuclear (three with SURF1). Overall, we identified mutations in 61% of LS patients (11/18 individuals) in this cohort. Sanger sequencing with our limited set of primers allowed us a rapid genetic confirmation of more than half of the LS patients and it appears to be efficient as a primary genetic screening in this cohort.

Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: Impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biery, B.J.; Stein, D.E.; Goodman, S.I.

The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span {approximately}7 kb. Fibroblast DNA from 64 unrelated glutaric academia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms found in 7 of its 11 exons. Several mutations were found in more than one patient, but no one prevalent mutation was detected in themore » general population. As expected from pedigree analysis, a single mutant allele causes GA1 in the Old Order Amish of Lancaster County, Pennsylvania. Several mutations have been expressed in Escherichia coli, and all produce diminished enzyme activity. Reduced activity in GCD encoded by the A421V mutation in the Amish may be due to impaired association of enzyme subunits. 13 refs., 5 figs., 3 tabs.« less

Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology.

PubMed

Yakushi, Toshiharu; Matsushita, Kazunobu

2010-05-01

Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) of acetic acid bacteria is a membrane-bound enzyme involved in the acetic acid fermentation by oxidizing ethanol to acetaldehyde coupling with reduction of membranous ubiquinone (Q), which is, in turn, re-oxidized by ubiquinol oxidase, reducing oxygen to water. PQQ-ADHs seem to have co-evolved with the organisms fitting to their own habitats. The enzyme consists of three subunits and has a pyrroloquinoline quinone, 4 heme c moieties, and a tightly bound Q as the electron transfer mediators. Biochemical, genetic, and electrochemical studies have revealed the unique properties of PQQ-ADH since it was purified in 1978. The enzyme is unique to have ubiquinol oxidation activity in addition to Q reduction. This mini-review focuses on the molecular properties of PQQ-ADH, such as the roles of the subunits and the cofactors, particularly in intramolecular electron transport of the enzyme from ethanol to Q. Also, we summarize biotechnological applications of PQQ-ADH as to enantiospecific oxidations for production of the valuable chemicals and bioelectrocatalysis for sensors and fuel cells using indirect and direct electron transfer technologies and discuss unsolved issues and future prospects related to this elaborate enzyme.

Acute high-altitude hypoxic brain injury: Identification of ten differential proteins

PubMed Central

Li, Jianyu; Qi, Yuting; Liu, Hui; Cui, Ying; Zhang, Li; Gong, Haiying; Li, Yaxiao; Li, Lingzhi; Zhang, Yongliang

2013-01-01

Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain injury. However, little is currently known about the mechanisms responsible for mitochondrial dysfunction in hypobaric hypoxic brain damage. In this study, a rat model of hypobaric hypoxic brain injury was established to investigate the molecular mechanisms associated with mitochondrial dysfunction. As revealed by two-dimensional electrophoresis analysis, 16, 21, and 36 differential protein spots in cerebral mitochondria were observed at 6, 12, and 24 hours post-hypobaric hypoxia, respectively. Furthermore, ten protein spots selected from each hypobaric hypoxia subgroup were similarly regulated and were identified by mass spectrometry. These detected proteins included dihydropyrimidinase-related protein 2, creatine kinase B-type, isovaleryl-CoA dehydrogenase, elongation factor Ts, ATP synthase beta-subunit, 3-mercaptopyruvate sulfurtransferase, electron transfer flavoprotein alpha-subunit, Chain A of 2-enoyl-CoA hydratase, NADH dehydrogenase iron-sulfur protein 8 and tropomyosin beta chain. These ten proteins are all involved in the electron transport chain and the function of ATP synthase. Our findings indicate that hypobaric hypoxia can induce the differential expression of several cerebral mitochondrial proteins, which are involved in the regulation of mitochondrial energy production. PMID:25206614

Is the c.3G>C mutation in the succinate dehydrogenase subunit D (SDHD) gene due to a founder effect in Chinese head and neck paraganglioma patients?

PubMed

Zha, Yang; Chen, Xing-ming; Lam, Ching-wan; Lee, Soo-chin; Tong, Sui-fan; Gao, Zhi-qiang

2011-08-01

Three Chinese patients with head and neck paragangliomas have been reported to carry the c.3G>C mutation in the succinate dehydrogenase subunit D (SDHD) gene. In addition, in our hospital, two further patients were identified who have the same mutation. It is unclear whether the c.3G>C mutation in Chinese patients is a recurrent mutation or if it is due to a founder effect. We conducted haplotype analysis on these patients to answer this question. Individual case-control study. Germ-line mutations were confirmed in the patients and their families examined in this study using direct sequencing. We also constructed and analyzed haplotypes in four Chinese families. Genotype frequencies were compared to the control group. Three of four families shared the same haplotype, which rarely occurred in the control group. The last family shared a very short area on the physical map with the other three families. There is a founder effect in Chinese head and neck paraganglioma patients carrying the SDHD c.3G>C mutation. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

Identification of a Novel HADHB Gene Mutation in an Iranian Patient with Mitochondrial Trifunctional Protein Deficiency.

PubMed

Shahrokhi, Mahdiyeh; Shafiei, Mohammad; Galehdari, Hamid; Shariati, Gholamreza

2017-01-01

Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of eight parts (subunits): four α-subunits containing LCEH (long-chain 2,3-enoyl-CoA  hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and four β-subunits that possess LCKT (long-chain  3-ketoacyl-CoA thiolase) activity which catalyzes three out of four steps in β-oxidation spiral of long-chain fatty acid. Its deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases. A blood spot was collected from the patient's original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS were performed. After isolation of DNA and Amplification of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the flanking introns both of genes were performed. Here, we report a novel mutation in a patient with MTP deficiency diagnosed with newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS and then confirmed by enzyme analysis in cultured fibroblasts and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation (p.Q385P) c.1154A > C in exon 14 of HADHB gene. Since this mutation was not found in 50 normal control cases; so it was concluded that c.1154A > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the MTP protein complex.

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I*

PubMed Central

Quinlan, Casey L.; Goncalves, Renata L. S.; Hey-Mogensen, Martin; Yadava, Nagendra; Bunik, Victoria I.; Brand, Martin D.

2014-01-01

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I. PMID:24515115

[Mitochondrial DNA genetic differentiation of the muksun Coregonus muksun (Pallas) and related Siberian species of Coregonus (Coredonidae, Salmoniformes)].

PubMed

Baldina, S N; Gordon, N Iu; Politov, D V

2008-07-01

Restriction enzyme analysis of the mitochondrial DNA (mtDNA) fragment encoding subunit 1 of the NADH dehydrogenase complex (ND-1) amplified via polymerase chain reaction (PCR) has been used to obtain data on genetic differentiation of muksun Coregonus muksun (Pallas) populations. Population polymorphism with respect to the restriction sites of 18 endonucleases has been described. It has been demonstrated that the muksun is genetically related to the pidschian C. pidschian (Gmelin), its sympatric species in Siberian waters. Analysis of the median network of mtDNA haplotypes has shown that haplotypes of muksun from various Siberian basins form a common group with haplotypes of pidschian of the Arctic Ocean basin, some frequent haplotypes been found in both forms. This raises the question as to the validity of the muksun as a species. Differences within this group of haplotypes are much smaller than those typical of species of the genus Coregonus. The possibility of a hybrid origin of the muksun from a pidschian-like ancestor and species of the cisco-peled (C. sardinella-C. peled) complex is discussed.

Dihydrolipoamide dehydrogenase-Lpd (Rv0462)-specific T cell recall responses are higher in healthy household contacts of TB: a novel immunodominant antigen from M. tuberculosis.

PubMed

Devasundaram, Santhi; Raja, Alamelu

2017-07-01

The partial effectiveness against pulmonary tuberculosis (PTB), displayed by the existing tuberculosis (TB) vaccine, bacillus Calmette-Guérin (BCG), highlights the need for novel vaccines to replace or improve BCG. In TB immunology, antigen-specific cellular immune response is frequently considered indispensable. Latency-associated antigens are intriguing as targets for TB vaccine development. The mycobacterial protein, dihydrolipoamide dehydrogenase (Lpd; Rv0462), the third enzyme of the pyruvate dehydrogenase (PDH) complex, facilitates Mycobacterium tuberculosis to resist host reactive nitrogen intermediates. Multicolor flow cytometry analysis of whole-blood cultures showed higher Lpd-specific Th1 recall response (IFN-γ, TNF-α, and IL-2; P = 0.0006) and memory CD4 + and CD8 + T cells (CCR7 + CD45RA - and CCR7 - CD45RA - ) in healthy household contacts (HHC) of TB ( P < 0.0001), which is comparable with or higher than the standard antigens, ESAT-6 and CFP-10. The frequency of Lpd-specific multifunctional T cells was higher in HHC compared with PTB patients. However, there is no significant statistical correlation. Regulatory T cell (T reg ) analysis of HHCs and active TB patients demonstrated very low Lpd-specific CD4 + T regs relative to ESAT-6 and CFP-10. Our study demonstrates that the Lpd antigen induces a strong cellular immune response in healthy mycobacteria-infected individuals. In consideration of this population having demonstrated immunologic protection against active TB disease development, our data are encouraging about the possible use of Lpd as a target for further TB subunit vaccine development. © Society for Leukocyte Biology.

Step-wise and lineage-specific diversification of plant RNA polymerase genes and origin of the largest plant-specific subunits.

PubMed

Wang, Yaqiong; Ma, Hong

2015-09-01

Proteins often function as complexes, yet little is known about the evolution of dissimilar subunits of complexes. DNA-directed RNA polymerases (RNAPs) are multisubunit complexes, with distinct eukaryotic types for different classes of transcripts. In addition to Pol I-III, common in eukaryotes, plants have Pol IV and V for epigenetic regulation. Some RNAP subunits are specific to one type, whereas other subunits are shared by multiple types. We have conducted extensive phylogenetic and sequence analyses, and have placed RNAP gene duplication events in land plant history, thereby reconstructing the subunit compositions of the novel RNAPs during land plant evolution. We found that Pol IV/V have experienced step-wise duplication and diversification of various subunits, with increasingly distinctive subunit compositions. Also, lineage-specific duplications have further increased RNAP complexity with distinct copies in different plant families and varying divergence for subunits of different RNAPs. Further, the largest subunits of Pol IV/V probably originated from a gene fusion in the ancestral land plants. We propose a framework of plant RNAP evolution, providing an excellent model for protein complex evolution. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Suppression of BRCA2 by Mutant Mitochondrial DNA in Prostate Cancer

DTIC Science & Technology

2011-05-01

Briefly, the electron transfer activities of complex I/III (NADH dehydrogenase/cytochrome bc1 complex: catalyzes the electron transfer from NADH to...ferricytochrome c) and complex II/III (succinate dehydrogenase/cytochrome bc1 complex: catalyzes the electron transfer from succinate to ferricytochrome...The electron transfer activity of complex IV (cytochrome c oxidase: catalyzes the final step of the respiratory chain by transferring electrons from

Overexpression, crystallization and preliminary X-­ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

PubMed Central

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-01-01

The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-­phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M) of 3.64 Å3 Da−1 and a solvent content of 66%. PMID:16511285

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I.

PubMed

Moparthi, Vamsi K; Kumar, Brijesh; Al-Eryani, Yusra; Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn; Hägerhäll, Cecilia

2014-01-01

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity. © 2013. Published by Elsevier B.V. All rights reserved.

Biogenesis of the yeast cytochrome bc1 complex.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Resolving the role of plant glutamate dehydrogenase: II. Physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously.

PubMed

Tercé-Laforgue, Thérèse; Bedu, Magali; Dargel-Grafin, Céline; Dubois, Frédéric; Gibon, Yves; Restivo, Francesco M; Hirel, Bertrand

2013-10-01

Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and β-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the mitochondria of the phloem companion cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

PubMed

Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-03-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

PubMed Central

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

PubMed

Tymecka-Mulik, Joanna; Boss, Lidia; Maciąg-Dorszyńska, Monika; Matias Rodrigues, João F; Gaffke, Lidia; Wosinski, Anna; Cech, Grzegorz M; Szalewska-Pałasz, Agnieszka; Węgrzyn, Grzegorz; Glinkowska, Monika

2017-01-01

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

PubMed

Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

2017-04-01

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pyruvate dehydrogenase expression is negatively associated with cell stemness and worse clinical outcome in prostate cancers

PubMed Central

Zhong, Yali; Li, Xiaoli; Ji, Yasai; Li, Xiaoran; Li, Yaqing; Yu, Dandan; Yuan, Yuan; Liu, Jian; Li, Huixiang; Zhang, Mingzhi; Ji, Zhenyu; Fan, Dandan; Wen, Jianguo; Goscinski, Mariusz Adam; Yuan, Long; Hao, Bin; Nesland, Jahn M; Suo, Zhenhe

2017-01-01

Cells generate adenosine-5′-triphosphate (ATP), the major currency for energy-consuming reactions, through mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. One of the remarkable features of cancer cells is aerobic glycolysis, also known as the “Warburg Effect”, in which cancer cells rely preferentially on glycolysis instead of mitochondrial OXPHOS as the main energy source even in the presence of high oxygen tension. One of the main players in controlling OXPHOS is the mitochondrial gatekeeperpyruvate dehydrogenase complex (PDHc) and its major subunit is E1α (PDHA1). To further analyze the function of PDHA1 in cancer cells, it was knock out (KO) in the human prostate cancer cell line LnCap and a stable KO cell line was established. We demonstrated that PDHA1 gene KO significantly decreased mitochondrial OXPHOS and promoted anaerobic glycolysis, accompanied with higher stemness phenotype including resistance to chemotherapy, enhanced migration ability and increased expression of cancer stem cell markers. We also examined PDHA1 protein expression in prostate cancer tissues by immunohistochemistry and observed that reduced PDHA1 protein expression in clinical prostate carcinomas was significantly correlated with poor prognosis. Collectively, our results show that negative PDHA1 gene expressionis associated with significantly higher cell stemness in prostate cancer cells and reduced protein expression of this gene is associated with shorter clinical outcome in prostate cancers. PMID:28076853

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

PubMed

Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B

2016-06-01

Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

PubMed

Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

2006-09-08

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits.

PubMed

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-07-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain‑dead donors. In the present study, two‑dimensional gel electrophoresis and MALDI‑TOF MS‑based comparative proteomic analysis were conducted to profile the differentially‑expressed proteins between brain death and the control group renal tissues. A total of 40 age‑ and sex‑matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two‑dimensional gel electrophoresis, >2‑fold alterations were identified by MALDI‑TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3‑N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b‑c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre‑mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V‑type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin‑3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time‑dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain‑dead donors.

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits

PubMed Central

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-01-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain-dead donors. In the present study, two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis were conducted to profile the differentially-expressed proteins between brain death and the control group renal tissues. A total of 40 age- and sex-matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two-dimensional gel electrophoresis, >2-fold alterations were identified by MALDI-TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3-N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b-c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre-mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V-type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin-3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time-dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain-dead donors. PMID:28534953

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).

PubMed

Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

2008-06-01

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design*

PubMed Central

Frayne, Jan; Taylor, Abby; Cameron, Gus; Hadfield, Andrea T.

2009-01-01

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the Ps pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives. PMID:19542219

Mitochondrial NADH Fluorescence is Enhanced by Complex I Binding

PubMed Central

Blinova, Ksenia; Levine, Rodney L.; Boja, Emily S.; Griffiths, Gary L.; Shi, Zhen-Dan; Ruddy, Brian; Balaban, Robert S.

2012-01-01

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state. PMID:18702505

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esclapez, Julia; Britton, K. Linda; Baker, Patrick J.

2005-08-01

Single crystals of binary and ternary complexes of wild-type and D38C mutant H. mediterranei glucose dehydrogenase have been obtained by the hanging-drop vapour-diffusion method. Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse themore » significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.« less

α-Lactalbumin-oleic acid complex kills tumor cells by inducing excess energy metabolism but inhibiting mRNA expression of the related enzymes.

PubMed

Fang, B; Zhang, M; Ge, K S; Xing, H Z; Ren, F Z

2018-06-01

Previous studies have demonstrated that the anti-tumor α-lactalbumin-oleic acid complex (α-LA-OA) may target the glycolysis of tumor cells. However, few data are available regarding the effects of α-LA-OA on energy metabolism. In this study, we measured glycolysis and mitochondrial functions in HeLa cells in response to α-LA-OA using the XF flux analyzer (Seahorse Bioscience, North Billerica, MA). The gene expression of enzymes involved in glycolysis, tricarboxylic acid cycle, electron transfer chain, and ATP synthesis were also evaluated. Our results show that α-LA-OA significantly enhanced the basal glycolysis and glycolytic capacity. Mitochondrial oxidative phosphorylation, including the basal respiration, maximal respiration, spare respiratory capacity and ATP production were also improved in response to α-LA-OA. The enhanced mitochondrial functions maybe partly due to the increased capacity of utilizing fatty acids and glutamine as the substrate. However, the gene expressions of pyruvate kinase M2, lactate dehydrogenase A, aconitate hydratase, and isocitrate dehydrogenase 1 were inhibited, suggesting an insufficient ability for the glycolysis process and the tricarboxylic acid cycle. The increased expression of acetyl-coenzyme A acyltransferase 2, a central enzyme involved in the β-oxidation of fatty acids, would enhance the unbalance due to the decreased expression of electron transfer flavoprotein β subunit, which acts as the electron acceptor. These results indicated that α-LA-OA may induce oxidative stress due to conditions in which the ATP production is exceeding the energy demand. Our results may help clarify the mechanism of apoptosis induced by reactive oxygen species and mitochondrial destruction. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Autoimmune liver serology: current diagnostic and clinical challenges.

PubMed

Bogdanos, Dimitrios-P; Invernizzi, Pietro; Mackay, Ian-R; Vergani, Diego

2008-06-07

Liver-related autoantibodies are crucial for the correct diagnosis and classification of autoimmune liver diseases (AiLD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children. AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA and anti-LKM antibodies can be present in de-novo AIH following liver transplantation. PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents as at least two variants, first the classical primary sclerosing cholangitis (PSC) mostly affecting adult men wherein the only (and non-specific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH. Liver diagnostic serology is a fast-expanding area of investigation as new purified and recombinant autoantigens, and automated technologies such as ELISAs and bead assays, become available to complement (or even compete with) traditional immunofluorescence procedures. We survey for the first time global trends in quality assurance impacting as it does on (1) manufacturers/purveyors of kits and reagents, (2) diagnostic service laboratories that fulfill clinicians' requirements, and (3) the end-user, the physician providing patient care, who must properly interpret test results in the overall clinical context.

Autoimmune liver serology: Current diagnostic and clinical challenges

PubMed Central

Bogdanos, Dimitrios P; Invernizzi, Pietro; Mackay, Ian R; Vergani, Diego

2008-01-01

Liver-related autoantibodies are crucial for the correct diagnosis and classification of autoimmune liver diseases (AiLD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children. AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA and anti-LKM antibodies can be present in de-novo AIH following liver transplantation. PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents as at least two variants, first the classical primary sclerosing cholangitis (PSC) mostly affecting adult men wherein the only (and non-specific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH. Liver diagnostic serology is a fast-expanding area of investigation as new purified and recombinant autoantigens, and automated technologies such as ELISAs and bead assays, become available to complement (or even compete with) traditional immunofluorescence procedures. We survey for the first time global trends in quality assurance impacting as it does on (1) manufacturers/purveyors of kits and reagents, (2) diagnostic service laboratories that fulfill clinicians’ requirements, and (3) the end-user, the physician providing patient care, who must properly interpret test results in the overall clinical context. PMID:18528935

Physiological and biochemical characterization of the soluble formate dehydrogenase, a molybdoenzyme from Alcaligenes eutrophus.

PubMed Central

Friedebold, J; Bowien, B

1993-01-01

Organoautotrophic growth of Alcaligenes eutrophus on formate was dependent on the presence of molybdate in the medium. Supplementation of the medium with tungstate lead to growth cessation. Corresponding effects of these anions were observed for the activity of the soluble, NAD(+)-linked formate dehydrogenase (S-FDH; EC 1.2.1.2) of the organism. Lack of molybdate or presence of tungstate resulted in an almost complete loss of S-FDH activity. S-FDH was purified to near homogeneity in the presence of nitrate as a stabilizing agent. The native enzyme exhibited an M(r) of 197,000 and a heterotetrameric quaternary structure with nonidentical subunits of M(r) 110,000 (alpha), 57,000 (beta), 19,400 (gamma), and 11,600 (delta). It contained 0.64 g-atom of molybdenum, 25 g-atom of nonheme iron, 20 g-atom of acid-labile sulfur, and 0.9 mol of flavin mononucleotide per mol. The fluorescence spectrum of iodine-oxidized S-FDH was nearly identical to the form A spectrum of milk xanthine oxidase, proving the presence of a pterin cofactor. The molybdenum-complexing cofactor was identified as molybdopterin guanine dinucleotide in an amount of 0.71 mol/mol of S-FDH. Apparent Km values of 3.3 mM for formate and 0.09 mM for NAD+ were determined. The enzyme coupled the oxidation of formate to a number of artificial electron acceptors and was strongly inactivated by formate in the absence of NAD+. It was inhibited by cyanide, azide, nitrate, and Hg2+ ions. Thus, the enzyme belongs to a new group of complex molybdo-flavo Fe-S FDH that so far has been detected in only one other aerobic bacterium. Images PMID:8335630

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

PubMed

Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

2016-01-01

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

(PS)2: protein structure prediction server version 3.0.

PubMed

Huang, Tsun-Tsao; Hwang, Jenn-Kang; Chen, Chu-Huang; Chu, Chih-Sheng; Lee, Chi-Wen; Chen, Chih-Chieh

2015-07-01

Protein complexes are involved in many biological processes. Examining coupling between subunits of a complex would be useful to understand the molecular basis of protein function. Here, our updated (PS)(2) web server predicts the three-dimensional structures of protein complexes based on comparative modeling; furthermore, this server examines the coupling between subunits of the predicted complex by combining structural and evolutionary considerations. The predicted complex structure could be indicated and visualized by Java-based 3D graphics viewers and the structural and evolutionary profiles are shown and compared chain-by-chain. For each subunit, considerations with or without the packing contribution of other subunits cause the differences in similarities between structural and evolutionary profiles, and these differences imply which form, complex or monomeric, is preferred in the biological condition for the subunit. We believe that the (PS)(2) server would be a useful tool for biologists who are interested not only in the structures of protein complexes but also in the coupling between subunits of the complexes. The (PS)(2) is freely available at http://ps2v3.life.nctu.edu.tw/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.

PubMed

Snyder, P M; Cheng, C; Prince, L S; Rogers, J C; Welsh, M J

1998-01-09

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements.

PubMed

Sahlan, Muhamad; Kanzaki, Taro; Zako, Tamotsu; Maeda, Mizuo; Yohda, Masafumi

2010-09-01

Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin beta subunit more strongly than the alpha subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnbeta subunits. Interestingly, chaperonin complexes containing two beta subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of beta subunits. The result suggests that all four beta tentacles of prefoldin interact with the helical protrusions of CPN in the PFD-CPN complex as the previously proposed model that two adjacent PFD beta subunits seem to interact with two CPN adjacent subunits. Copyright © 2010 Elsevier B.V. All rights reserved.

The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

PubMed

Yang, Guowei; Waterfield, Nicholas R

2013-01-01

The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification.

PubMed

Lawlis, V B; Roche, T E

1980-11-20

NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex was compared at 10 microM free Ca2+ or in the absence of Ca2+ (i.e., less than 1.0 nM free Ca2+). In the presence of Ca2+, NADH inhibition was appreciably decreased for a wide range of NADH:NAD+ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 microM free Ca/+ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca2+ on the S0.5 for alpha-ketoglutarate which was decreased by Ca2+ with a half-maximal effect at a similar concentration. The effect of Ca2+ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex is required to elicit the effect of Ca2+ on NADH inhibition. At a fixed alpha-ketoglutarate concentration (50 microM), removal of Ca2+ reduced the activity of the alpha-ketoglutarate dehydrogenase complex by 8.5-fold (due to an increase in S0.5 for alpha-ketoglutarate) and, in the presence of different NADH:NAD+ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxPi) or a combination of the phosphate potential and NADH:NAD+ ratio are also described. The possibility that the level of intramitochondrial free Ca/+ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.

Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases*

PubMed Central

Nagel, Zachary D.; Cun, Shujian; Klinman, Judith P.

2013-01-01

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases. PMID:23525111

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat.

PubMed Central

Tsuji, S; Qureshi, M A; Hou, E W; Fitch, W M; Li, S S

1994-01-01

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously. Images PMID:7937776

ENU mutagenesis identifies mice with cardiac fibrosis and hepatic steatosis caused by a mutation in the mitochondrial trifunctional protein beta-subunit.

PubMed

Kao, Hsiao-Jung; Cheng, Ching-Feng; Chen, Yen-Hui; Hung, Shuen-Iu; Huang, Cheng-Chih; Millington, David; Kikuchi, Tateki; Wu, Jer-Yuarn; Chen, Yuan-Tsong

2006-12-15

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

PubMed

Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

2015-04-03

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

PubMed

Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

2017-07-01

The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

PubMed

Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

2015-03-01

The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein Flexibility Facilitates Quaternary Structure Assembly and Evolution

PubMed Central

Marsh, Joseph A.; Teichmann, Sarah A.

2014-01-01

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components. PMID:24866000

PDH E1β deficiency with novel mutations in two patients with Leigh syndrome.

PubMed

Quintana, E; Mayr, J A; García Silva, M T; Font, A; Tortoledo, M A; Moliner, S; Ozaez, L; Lluch, M; Cabello, A; Ricoy, J R; Koch, J; Ribes, A; Sperl, W; Briones, P

2009-12-01

Most cases of pyruvate dehydrogenase complex (PDHc) deficiency are attributable to mutations in the PDHA1 gene which encodes the E(1)α subunit, with few cases of mutations in the genes for E(3), E3BP (E(3) binding protein), E(2) and E(1)-phosphatase being reported. Only seven patients with deficiency of the E(1)β subunit have been described, with mutations in the PDHB gene in six of them. Clinically they presented with a non-specific encephalomyopathy. We report two patients with new mutations in PDHB and Leigh syndrome. Patient 1 was a boy with neonatal onset of hyperlactataemia, corpus callosum hypoplasia and a convulsive encephalopathy. After neurological deterioration, he died at age 5 months. Autopsy revealed the characteristic features of Leigh syndrome. Patient 2, also a boy, presented a milder clinical course. First symptoms were noticed at age 16 months with muscular hypotonia, lactic acidosis and recurrent episodes of somnolence and transient tetraparesis. MRI revealed bilateral signal hyperintensities in the globus pallidus, midbrain and crura cerebri. PDHc and E(1) activities were deficient in fibroblasts in patient 1; in patient 2 PDHc deficiency was found in skeletal muscle. Mutations in PDHA1 were excluded. Sequencing of PDHB revealed a homozygous point mutation (c.302T>C), causing a predicted amino acid change (p.M101T) in patient 1. Patient 2 is compound heterozygote for mutations c.301A>G (p.M101V) and c.313G>A (p.R105Q). All three mutations appear to destabilize the E(1) enzyme with a decrease of both E(1)α and E(1)β subunits in immunoblot analysis. To our knowledge, these patients with novel PDHB mutations are the first reported with Leigh syndrome.

Stabilization of non-productive conformations underpins rapid electron transfer to electron-transferring flavoprotein.

PubMed

Toogood, Helen S; van Thiel, Adam; Scrutton, Nigel S; Leys, David

2005-08-26

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.

Enhanced Hydrogen Production from Formic Acid by Formate Hydrogen Lyase-Overexpressing Escherichia coli Strains

PubMed Central

Yoshida, Akihito; Nishimura, Taku; Kawaguchi, Hideo; Inui, Masayuki; Yukawa, Hideaki

2005-01-01

Genetic recombination of Escherichia coli in conjunction with process manipulation was employed to elevate the efficiency of hydrogen production in the resultant strain SR13 2 orders of magnitude above that of conventional methods. The formate hydrogen lyase (FHL)-overexpressing strain SR13 was constructed by combining FHL repressor (hycA) inactivation with FHL activator (fhlA) overexpression. Transcription of large-subunit formate dehydrogenase, fdhF, and large-subunit hydrogenase, hycE, in strain SR13 increased 6.5- and 7.0-fold, respectively, compared to the wild-type strain. On its own, this genetic modification effectively resulted in a 2.8-fold increase in hydrogen productivity of SR13 compared to the wild-type strain. Further enhancement of productivity was attained by using a novel method involving the induction of the FHL complex with high-cell-density filling of a reactor under anaerobic conditions. Continuous hydrogen production was achieved by maintaining the reactor concentration of the substrate (free formic acid) under 25 mM. An initial productivity of 23.6 g hydrogen h−1 liter−1 (300 liters h−1 liter−1 at 37°C) was achieved using strain SR13 at a cell density of 93 g (dry weight) cells/liter. The hydrogen productivity reported in this work has great potential for practical application. PMID:16269707

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ming; Smith, Christopher J.; Walker, Matthew T.

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shownmore » here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.« less

The succinate dehydrogenase assembly factor, SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria.

PubMed

McNeil, Matthew B; Hampton, Hannah G; Hards, Kiel J; Watson, Bridget N J; Cook, Gregory M; Fineran, Peter C

2014-01-31

The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Assignment of Etfdh, Etfb, and Etfa to chromosomes 3, 7, and 13: The mouse homologs of genes respondible for glutaric acidemia type II in human

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, R.A.; Dowler, L.L.; Angeloni, S.V.

Electron transfer flavoprotein (composed of {alpha} and {beta} subunits) is an obligatory electron acceptor for several dehydrogenases and is located in the mitochondrial matrix. Electrons accepted by electron transfer flavo-protein (ETF) are transferred to the main mitochondrial respiratory chain by the way of ETF dehydrogenase (ETFDH). In humans, deficiency of ETF or ETFDH leads to glutaric acidemia type II, an inherited metabolic disorder that can be fatal in its neonatal form and is characterized by severe hypoketotic hypoglycemia and acidosis. We used cDNA probes for the Etfdh, Etfb, and Etfa genes to determine localization of these mouse genes to chromosomesmore » 3, 7, and 13. 18 refs., 3 figs.« less

Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans.

PubMed

Fitzgerald, P M; Duax, W L; Punzi, J S; Orr, J C

1984-05-15

3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.

Structural changes of homodimers in the PDB.

PubMed

Koike, Ryotaro; Amemiya, Takayuki; Horii, Tatsuya; Ota, Motonori

2018-04-01

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome.

PubMed

Yu, Zanlin; Kleifeld, Oded; Lande-Atir, Avigail; Bsoul, Maisa; Kleiman, Maya; Krutauz, Daria; Book, Adam; Vierstra, Richard D; Hofmann, Kay; Reis, Noa; Glickman, Michael H; Pick, Elah

2011-04-01

Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.

Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex*

PubMed Central

Setiaputra, Dheva; Ross, James D.; Lu, Shan; Cheng, Derrick T.; Dong, Meng-Qiu; Yip, Calvin K.

2015-01-01

The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

Distribution of the Pyruvate Dehydrogenase Complex in Developing Soybean Cotyledons

USDA-ARS?s Scientific Manuscript database

The somewhat surprising report that storage proteins and oil are non-uniformly distributed in the cotyledons of developing soybeans prompted us to determine the spatial distribution of the mitochondrial and plastidial forms of the pyruvate dehydrogenase complex (PDC). It has been proposed that pla...

Prevalence and genetic characterization of Cryptosporidium species and Giardia duodenalis in lambs in Oromia Special Zone, Central Ethiopia.

PubMed

Wegayehu, Teklu; Karim, Md Robiul; Li, Junqiang; Adamu, Haileeyesus; Erko, Berhanu; Zhang, Longxian; Tilahun, Getachew

2017-01-17

Cryptosporidium and Giardia duodenalis are gastro-intestinal parasites that infect human and animals worldwide. Both parasites share a broad host range and are believed to be zoonosis. The aim of this study was to identify the species of Cryptosporidium and assemblages of G. duodenalis in lambs and to elucidate their role in zoonotic transmission. A total of 389 fecal samples were collected from lambs and screened by microscopy and nested PCR targeting the small-subunit ribosomal RNA for Cryptosporidium; and the small-subunit ribosomal RNA, triose phosphate isomerase, β-giardin, and glutamate dehydrogenase genes for G. duodenalis. The prevalence of Cryptosporidium and G. duodenalis was 2.1% (8/389) and 2.6% (10/389), respectively. The infection rate at the three study sites ranged from 1.3 to 3.1% for Cryptosporidium and 1.6 to 3.9% for G. duodenalis; but variation was not statistically significant (p > 0.05). The finding also showed that there is no sex and age group associated difference in the occurrence of Cryptosporidium and G. duodenalis infections in lambs. Sequence analysis revealed that lambs were mono-infection with C. ubiquitum and G. duodenalis assemblage E. The analysis also indicated the presence of genetic variation within isolates of assemblage E; with 4 of them are novel genotypes at the small-subunit ribosomal RNA, β-giardin, and glutamate dehydrogenase genes. The findings of the current study showed that lambs are capable of harboring C. ubiquitum and G. duodenalis assemblage E. This finding suggests that lambs might be sources for potentially zoonotic Cryptosporidium species. This was first molecular study in lambs and contributes to a better understanding of the epidemiology of Cryptosporidium and G. duodenalis in central Ethiopia.

Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil▿

PubMed Central

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-01-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-08-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

A novel mitochondrial genome of Arborophila and new insight into Arborophila evolutionary history.

PubMed

Yan, Chaochao; Mou, Biqin; Meng, Yang; Tu, Feiyun; Fan, Zhenxin; Price, Megan; Yue, Bisong; Zhang, Xiuyue

2017-01-01

The lineage of the Bar-backed Partridge (Arborophila brunneopectus) was investigated to determine the phylogenetic relationships within Arborophila as the species is centrally distributed within an area covered by the distributions of 22 South-east Asian hill partridge species. The complete mitochondrial genome (mitogenome) of A. brunneopectus was determined and compared with four other hill partridge species mitogenomes. NADH subunit genes are radical in hill partridge mitogenomes and contain the most potential positive selective sites around where variable sites are abundant. Together with 44 other mitogenomes of closely related species, we reconstructed highly resolved phylogenetic trees using maximum likelihood (ML) and Bayesian inference (BI) analyses and calculated the divergence and dispersal history of Arborophila using combined datasets composed of their 13-protein coding sequences. Arborophila is reportedly be the oldest group in Phasianidae whose ancestors probably originated in Asia. A. rufipectus shares a closer relationship with A. ardens and A. brunneopectus compared to A. gingica and A. rufogularis, and such relationships were supported and profiled by NADH dehydrogenase subunit 5 (ND5). The intragenus divergence of all five Arborophila species occurred in the Miocene (16.84~5.69 Mya) when there were periods of climate cooling. We propose that these cooling events in the Miocene forced hill partridges from higher to lower altitudes, which led to geographic isolation and speciation. We demonstrated that the apparently deleterious +1 frameshift mutation in NADH dehydrogenase subunit 3 (ND3) found in all Arborophila is an ancient trait that has been eliminated in some younger lineages, such as Passeriformes. It is unclear of the biological advantages of this elimination for the relevant taxa and this requires further investigation.

A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate.

PubMed

Fokina, K V; Dainyak, M B; Nagradova, N K; Muronetz, V I

1997-09-15

The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

The adaptive evolution of the mammalian mitochondrial genome

PubMed Central

da Fonseca, Rute R; Johnson, Warren E; O'Brien, Stephen J; Ramos, Maria João; Antunes, Agostinho

2008-01-01

Background The mitochondria produce up to 95% of a eukaryotic cell's energy through oxidative phosphorylation. The proteins involved in this vital process are under high functional constraints. However, metabolic requirements vary across species, potentially modifying selective pressures. We evaluate the adaptive evolution of 12 protein-coding mitochondrial genes in 41 placental mammalian species by assessing amino acid sequence variation and exploring the functional implications of observed variation in secondary and tertiary protein structures. Results Wide variation in the properties of amino acids were observed at functionally important regions of cytochrome b in species with more-specialized metabolic requirements (such as adaptation to low energy diet or large body size, such as in elephant, dugong, sloth, and pangolin, and adaptation to unusual oxygen requirements, for example diving in cetaceans, flying in bats, and living at high altitudes in alpacas). Signatures of adaptive variation in the NADH dehydrogenase complex were restricted to the loop regions of the transmembrane units which likely function as protons pumps. Evidence of adaptive variation in the cytochrome c oxidase complex was observed mostly at the interface between the mitochondrial and nuclear-encoded subunits, perhaps evidence of co-evolution. The ATP8 subunit, which has an important role in the assembly of F0, exhibited the highest signal of adaptive variation. ATP6, which has an essential role in rotor performance, showed a high adaptive variation in predicted loop areas. Conclusion Our study provides insight into the adaptive evolution of the mtDNA genome in mammals and its implications for the molecular mechanism of oxidative phosphorylation. We present a framework for future experimental characterization of the impact of specific mutations in the function, physiology, and interactions of the mtDNA encoded proteins involved in oxidative phosphorylation. PMID:18318906

Identification of mutations, genotype-phenotype correlation and prenatal diagnosis of maple syrup urine disease in Indian patients.

PubMed

Gupta, Deepti; Bijarnia-Mahay, Sunita; Saxena, Renu; Kohli, Sudha; Dua-Puri, Ratna; Verma, Jyotsna; Thomas, E; Shigematsu, Yosuke; Yamaguchi, Seiji; Deb, Roumi; Verma, Ishwar Chander

2015-09-01

Maple syrup urine disease (MSUD) is caused by mutations in genes BCKDHA, BCKDHB, DBT encoding E1α, E1β, and E2 subunits of enzyme complex, branched-chain alpha-ketoacid dehydrogenase (BCKDH). BCKDH participates in catabolism of branched-chain amino acids (BCAAs) - leucine, isoleucine and valine in the energy production pathway. Deficiency or defect in the enzyme complex causes accumulation of BCAAs and keto-acids leading to toxicity. Twenty-four patients with MSUD were enrolled in the study for molecular characterization and genotype-phenotype correlation. Molecular studies were carried out by sequencing of the 3 genes by Sanger method. Bioinformatics tools were employed to classify novel variations into pathogenic or benign. The predicted effects of novel changes on protein structure were elucidated by 3D modeling. Mutations were detected in 22 of 24 patients (11, 7 and 4 in BCKDHB, BCKDHA and DBT genes, respectively). Twenty mutations including 11 novel mutations were identified. Protein modeling in novel mutations showed alteration of structure and function of these subunits. Mutations, c.1065 delT (BCKDHB gene) and c.939G > C (DBT gene) were noted to be recurrent, identified in 6 of 22 alleles and 5 of 8 alleles, respectively. Two-third patients were of neonatal classical phenotype (16 of 24). BCKDHB gene mutations were present in 10 of these 16 patients. Prenatal diagnoses were performed in 4 families. Consanguinity was noted in 37.5% families. Although no obvious genotype-phenotype correlation could be found in our study, most cases with mutation in BCKDHB gene presented in neonatal period. Large number of novel mutations underlines the heterogeneity and distinctness of gene pool from India. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Analysis of a Range of Catabolic Mutants Provides Evidence That Phytanoyl-Coenzyme A Does Not Act as a Substrate of the Electron-Transfer Flavoprotein/Electron-Transfer Flavoprotein:Ubiquinone Oxidoreductase Complex in Arabidopsis during Dark-Induced Senescence1[W][OA

PubMed Central

Araújo, Wagner L.; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Tohge, Takayuki; Larson, Tony R.; Krahnert, Ina; Balbo, Ilse; Witt, Sandra; Dörmann, Peter; Graham, Ian A.; Leaver, Christopher J.; Fernie, Alisdair R.

2011-01-01

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex. PMID:21788362

Neurospora crassa alpha-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties.

PubMed

Bessam, H; Mareck, A M; Foucher, B

1989-01-27

A method is proposed for the purification of the Neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-polyacrylamide gel electrophoresis as E3, E1 and E2 with respective Mr values of 54,000, 53,000 and 49,000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca2+ in particular was investigated. It was shown that Ca2+ diminishes the Km value of the N. crassa alpha-ketoglutarate dehydrogenase complex for alpha-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.

Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui†

PubMed Central

Johnsen, Ulrike; Schönheit, Peter

2004-01-01

During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui. PMID:15342590

Proteomic changes in Corbicula fluminea exposed to wastewater from a psychiatric hospital.

PubMed

Bebianno, M J; Sroda, S; Gomes, T; Chan, P; Bonnafe, E; Budzinski, H; Geret, F

2016-03-01

The increase use of pharmaceutical compounds in veterinary practice and human population results in the ubiquitous presence of these compounds in aquatic ecosystems. Because pharmaceuticals are highly bioactive, there is concern about their toxicological effects in aquatic organisms. Therefore, the aim of this study was to assess the effects of an effluent from a psychiatric hospital (containing a complex mixture of 25 pharmaceutical compounds from eleven therapeutic classes) on the freshwater clam Corbicula fluminea using a proteomic approach. The exposure of C. fluminea to this complex effluent containing anxiolytics, analgesics, lipid regulators, beta blockers, antidepressants, antiepileptics, antihistamines, antihypertensives, antiplatelets and antiarrhythmics induced protein changes after 1 day of exposure in clam gills and digestive gland more evident in the digestive gland. These changes included increase in the abundance of proteins associated with structural (actin and tubulin), cellular functions (calreticulin, proliferating cell nuclear antigen (PCNA), T complex protein 1 (TCP1)) and metabolism (aldehyde dehydrogenase (ALDH), alcohol dehydrogenase, 6 phosphogluconate dehydrogenase). Results from this study indicate that calreticulin, PCNA, ALDH and alcohol dehydrogenase in the digestive gland and T complex protein 1 (TCP1)) and 6 phosphogluconate dehydrogenase in the gills represent useful biomarkers for the ecotoxicological characterization of psychiatric hospital effluents in this species.

Two redundant octanoyltransferases and one obligatory lipoyl synthase provide protein-lipoylation autonomy to plastids of Arabidopsis.

PubMed

Ewald, R; Hoffmann, C; Neuhaus, E; Bauwe, H

2014-01-01

Octanoyltransferases (LIP2) are important for the lipoylation of several α-ketoacid decarboxylases and glycine decarboxylase, all of which are essential multienzyme complexes of central metabolism, by attaching de novo-synthesised octanoyl moieties to the respective target subunits. Lipoyl synthase (LIP1) then inserts two sulphur atoms each into the protein-bound octanoyl chains to generate the functional lipoamide arms. In plants, most of the above multienzyme complexes occur only in mitochondria. Pyruvate dehydrogenase is an exception, since it also occurs in plastids. Plastidial LIP1 and LIP2 are known, but it is not clear how essential these enzymes are. Here, we report that not just one but two redundant LIP2 isoforms, LIP2p and LIP2p2, operate in plastids of Arabidopsis. The combined deletion of the two isoenzymes is embryo-lethal. Deletion of the plastidial lipoyl synthase LIP1p is also embryo-lethal, indicating that all plastidial LIP1 activity is due to LIP1p. These features suggest that protein lipoylation is based on an autonomous and partially redundant de novo lipoylation pathway in plastids. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

USDA-ARS?s Scientific Manuscript database

NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

Evolution of disorder in Mediator complex and its functional relevance

PubMed Central

Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K.

2016-01-01

Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of ‘junction-MoRF’ has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein–protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms. PMID:26590257

The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate.

PubMed

Nemeria, Natalia S; Gerfen, Gary; Guevara, Elena; Nareddy, Pradeep Reddy; Szostak, Michal; Jordan, Frank

2017-07-01

Recently, we reported that the human 2-oxoglutarate dehydrogenase (hE1o) component of the 2-oxoglutarate dehydrogenase complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18

Expression of regulatory proteins in choroid plexus changes in early stages of Alzheimer disease.

PubMed

Krzyzanowska, Agnieszka; García-Consuegra, Inés; Pascual, Consuelo; Antequera, Desiree; Ferrer, Isidro; Carro, Eva

2015-04-01

Recent studies indicate that the choroid plexus has important physiologic and pathologic roles in Alzheimer disease (AD). To obtain additional insight on choroid plexus function, we performed a proteomic analysis of choroid plexus samples from patients with AD stages I to II (n = 16), III to IV (n = 16), and V to VI (n = 11) and 7 age-matched control subjects. We used 2-dimensional differential gel electrophoresis coupled with mass spectrometry to generate a complete picture of changes in choroid plexus protein expression occurring in AD patients. We identified 6 proteins: 14-3-3 β/α, 14-3-3 ε, moesin, proteasome activator complex subunit 1, annexin V, and aldehyde dehydrogenase, which were significantly regulated in AD patient samples (p < 0.05, >1.5-fold variation in expression vs control samples). These proteins are implicated in major physiologic functions including mitochondrial dysfunction and apoptosis regulation. These findings contribute additional significance to the emerging importance of molecular and functional changes of choroid plexus function in the pathophysiology of AD.

Respiratory processes in non-photosynthetic plastids

PubMed Central

Renato, Marta; Boronat, Albert; Azcón-Bieto, Joaquín

2015-01-01

Chlororespiration is a respiratory process located in chloroplast thylakoids which consists in an electron transport chain from NAD(P)H to oxygen. This respiratory chain involves the NAD(P)H dehydrogenase complex, the plastoquinone pool and the plastid terminal oxidase (PTOX), and it probably acts as a safety valve to prevent the over-reduction of the photosynthetic machinery in stress conditions. The existence of a similar respiratory activity in non-photosynthetic plastids has been less studied. Recently, it has been reported that tomato fruit chromoplasts present an oxygen consumption activity linked to ATP synthesis. Etioplasts and amyloplasts contain several electron carriers and some subunits of the ATP synthase, so they could harbor a similar respiratory process. This review provides an update on the study about respiratory processes in chromoplasts, identifying the major gaps that need to be addressed in future research. It also reviews the proteomic data of etioplasts and amyloplasts, which suggest the presence of a respiratory electron transport chain in these plastids. PMID:26236317

Juvenile Leigh syndrome, optic atrophy, ataxia, dystonia, and epilepsy due to T14487C mutation in the mtDNA-ND6 gene: a mitochondrial syndrome presenting from birth to adolescence.

PubMed

Leshinsky-Silver, Esther; Shuvalov, Ruslan; Inbar, Shani; Cohen, Sarit; Lev, Dorit; Lerman-Sagie, Tally

2011-04-01

An increasing number of reports describe mutations in mitochondrial DNA coding regions, especially in mitochondrial DNA- encoded nicotinamide adenine dinucleotide dehydrogenase subunit genes of the respiratory chain complex I, as causing early-onset Leigh syndrome. The authors report the molecular findings in a 24-year-old patient with juvenile-onset Leigh syndrome presenting with optic atrophy, ataxia dystonia, and epilepsy. A brain magnetic resonance imaging revealed bilateral basal ganglia and thalamic hypointensities, and a magnetic resonance spectroscopy revealed an increased lactate peak. The authors identified a T14487C change causing M63V substitution in the mitochondrial ND6 gene. The mutation was heteroplasmic in muscle and blood samples, with different mutation loads, and was absent in the patient's mother's urine and blood samples. They suggest that the T14487C mtDNA mutation should be analyzed in Leigh syndrome, presenting with optic atrophy, ataxia, dystonia, and epilepsy, regardless of age.

Cellulose microfibril structure: inspirations from plant diversity

NASA Astrophysics Data System (ADS)

Roberts, A. W.

2018-03-01

Cellulose microfibrils are synthesized at the plasma membrane by cellulose synthase catalytic subunits that associate to form cellulose synthesis complexes. Variation in the organization of these complexes underlies the variation in cellulose microfibril structure among diverse organisms. However, little is known about how the catalytic subunits interact to form complexes with different morphologies. We are using an evolutionary approach to investigate the roles of different catalytic subunit isoforms in organisms that have rosette-type cellulose synthesis complexes.

Integrative Approach for Computationally Inferring Interactions between the Alpha and Beta Subunits of the Calcium-Activated Potassium Channel (BK): a Docking Study

PubMed Central

González, Janneth; Gálvez, Angela; Morales, Ludis; Barreto, George E.; Capani, Francisco; Sierra, Omar; Torres, Yolima

2013-01-01

Three-dimensional models of the alpha- and beta-1 subunits of the calcium-activated potassium channel (BK) were predicted by threading modeling. A recursive approach comprising of sequence alignment and model building based on three templates was used to build these models, with the refinement of non-conserved regions carried out using threading techniques. The complex formed by the subunits was studied by means of docking techniques, using 3D models of the two subunits, and an approach based on rigid-body structures. Structural effects of the complex were analyzed with respect to hydrogen-bond interactions and binding-energy calculations. Potential interaction sites of the complex were determined by referencing a study of the difference accessible surface area (DASA) of the protein subunits in the complex. PMID:23492851

First Report of Echinococcus equinus in a Donkey in Turkey

PubMed Central

Simsek, Sami; Roinioti, Erifylli; Eroksuz, Hatice

2015-01-01

A 2-year-old female donkey (Equus asinus) was euthanized in the Pathology Department of Firat University, Elazig, Turkey. Necropsy disclosed the presence of 7 hydatid cysts distributed throughout the lung parenchyma. One of those cysts represented the parasite material of the present study and was molecularly identified through sequencing of a fragment of cytochrome c oxidase subunit 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (NADH1) gene, as Echinococcus equinus. The generated CO1 sequence supports the presence of the dominant haplotype as has been described in Europe and Africa. The NADH1 sequence was found similar to sequences reported in equids in Egypt and the United Kingdom. The molecular identification of E. equinus in a donkey is being reported for the first time in Turkey. PMID:26797441

First Report of Echinococcus equinus in a Donkey in Turkey.

PubMed

Simsek, Sami; Roinioti, Erifylli; Eroksuz, Hatice

2015-12-01

A 2-year-old female donkey (Equus asinus) was euthanized in the Pathology Department of Firat University, Elazig, Turkey. Necropsy disclosed the presence of 7 hydatid cysts distributed throughout the lung parenchyma. One of those cysts represented the parasite material of the present study and was molecularly identified through sequencing of a fragment of cytochrome c oxidase subunit 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (NADH1) gene, as Echinococcus equinus. The generated CO1 sequence supports the presence of the dominant haplotype as has been described in Europe and Africa. The NADH1 sequence was found similar to sequences reported in equids in Egypt and the United Kingdom. The molecular identification of E. equinus in a donkey is being reported for the first time in Turkey.

High prevalence of carriers of variant c.1528G>C of HADHA gene causing long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the population of adult Kashubians from North Poland.

PubMed

Nedoszytko, Bogusław; Siemińska, Alicja; Strapagiel, Dominik; Dąbrowski, Sławomir; Słomka, Marcin; Sobalska-Kwapis, Marta; Marciniak, Błażej; Wierzba, Jolanta; Skokowski, Jarosław; Fijałkowski, Marcin; Nowicki, Roman; Kalinowski, Leszek

2017-01-01

The mitochondrial β-oxidation of fatty acids is a complex catabolic pathway. One of the enzymes of this pathway is the heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits. Mutations in MTP genes (HADHA and HADHB), both located on chromosome 2p23, cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by decreased activity of MTP. The most common MTP mutation is long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency caused by the c.1528G>C (rs137852769, p.Glu510Gln) substitution in exon 15 of the HADHA gene. We analyzed the frequency of genetic variants in the HADHA gene in the adults of Kashubian origin from North Poland and compared this data in other Polish provinces. We found a significantly higher frequency of HDHA c.1528G>C (rs137852769, p.Glu510Gln) carriers among Kashubians (1/57) compared to subjects from other regions of Poland (1/187). We found higher frequency of c.652G>C (rs71441018, pVal218Leu) polymorphism in the HADHA gene within population of Silesia, southern Poland (1/107) compared to other regions. Our study indicate described high frequency of c.1528G>C variant of HADHA gene in Kashubian population, suggesting the founder effect. For the first time we have found high frequency of rs71441018 in the South Poland Silesian population.

DNA Carrier Testing and Newborn Screening for Maple Syrup Urine Disease in Old Order Mennonite Communities

PubMed Central

Carleton, Stephanie M.; Peck, Dawn S.; Grasela, Julie; Dietiker, Kristin L.

2010-01-01

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by mutations in the branched chain α-keto acid dehydrogenase complex. Worldwide incidence of MSUD is 1:225,000 live births. However, within Old Order Mennonite communities, the incidence is 1:150 live births and results from a common tyrosine to asparagine substitution (Y438N) in the E1α subunit of branched chain α-keto acid dehydrogenase. We developed a new DNA diagnostic assay utilizing TaqMan® technology and compared its efficacy, sensitivity, and duration with an existing polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. Carrier testing was performed by both TaqMan technology and PCR-RFLP on DNA isolated from buccal swabs of 160 individuals as well as from buccal swabs and blood spots of nine at-risk newborns; assay time, sensitivity, and reliability were also evaluated. The TaqMan assay, like the PCR-RFLP assay, accurately determined Y438N E1α allele status. However, the TaqMan assay appeared (1) more sensitive than the PCR-RFLP assay, requiring 10-fold less DNA (10 ng) to reliably determine genotype status and (2) faster, reducing the assay time required for diagnosis from ∼12 to 5 h. TaqMan technology allowed more rapid DNA diagnoses of MSUD in the neonate, thereby reducing the likelihood of neurological impairment while enhancing health and prognosis for affected infants. PMID:20136525

The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes.

PubMed

Batista, Ana P; Marreiros, Bruno C; Pereira, Manuela M

2013-05-01

We have recently investigated the long-recognized relationship between complex I and group 4 [NiFe] hydrogenases and we have established the so-called Energy-converting hydrogenase related (Ehr) complex as a new member of the family. We have also observed that four subunits, homologues to NuoB, D, H and L, are common to the members of the family. We have designated this common group of subunits the universal adaptor. Taking into account the similarity of the Na(+)/H(+) antiporter-like subunits of complex I (NuoL, NuoM and NuoN) and the unique structural characteristic of the long amphipathic α helix part of NuoL, the nature of the antiporter-like subunit of the universal adaptor was questioned. Thus, in this work we further explore the properties of the universal adaptor, investigating which antiporter-like subunit is part of the universal adaptor. We observe that the universal adaptor contains an antiporter-like subunit with a long amphipathic α helix, similar to NuoL. Consequently, the long helix is a common denominator that has been conserved in all members of the family. Such conservation surely reflects the key role of such helix in the energy transduction mechanism of this family of enzymes.

Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

PubMed

Repizo, Guillermo D; Blancato, Víctor S; Mortera, Pablo; Lolkema, Juke S; Magni, Christian

2013-05-01

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

USDA-ARS?s Scientific Manuscript database

Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES

DTIC Science & Technology

bound lipoic acid and 17 moles of bound FAD. Alpha -ketoglutarate dehydrogenase complex contains approximately 10 moles of protein-bound lipoic acid , 9...typical metal activators of oxidative decarboxylation reaction of alpha -keto acid . These activating effects were in good agreement with the results of...A coenzyme A- and NAD-linked pyruvate and alpha -ketoglutarate dehydrogenase complexes have been isolated from pig heart muscle as multienzyme units

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

PubMed Central

Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

2013-01-01

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria 1

PubMed Central

Chauveau, Michèle; Lance, Claude

1991-01-01

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca2+ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH. Images Figure 1 Figure 3 Figure 7 PMID:16668075

Limonoate dehydrogenase from Arthrobacter globiformis: the native enzyme and its N-terminal sequence.

PubMed

Suhayda, C G; Omura, M; Hasegawa, S

1995-09-01

Bitter limonoids in citrus juice lower the quality and value of commercial juices. Limonoate dehydrogenase converts the precursor of bitter limonin, limonoate A-ring lactone, to nonbitter 17-dehydrolimonoate A-ring lactone. This enzyme was isolated from Arthrobacter globiformis cells by a combination of ammonium sulfate fractionation, Cibacron Blue affinity chromatography and DEAE ion exchange HPLC. Using this protocol a 428-fold purification of the enzyme was obtained. Gel filtration HPLC indicated a M(r) of 118,000 for the native enzyme. SDS-PAGE indicated an individual subunit M(r) of 31,000. N-Terminal sequencing of the protein provided a sequence of the first 16 amino acid residues. Since LDH activity in citrus is very low, cloning the gene for this bacterial enzyme into citrus trees should enhance the natural debittering mechanism in citrus fruit.

Molecular architecture of the TRAPPII complex and implications for vesicle tethering.

PubMed

Yip, Calvin K; Berscheminski, Julia; Walz, Thomas

2010-11-01

Multisubunit tethering complexes participate in the process of vesicle tethering--the initial interaction between transport vesicles and their acceptor compartments. TRAPPII (named for transport protein particle II) is a highly conserved tethering complex that functions in the late Golgi apparatus and consists of all of the subunits of TRAPPI and three additional, specific subunits. We have purified native yeast TRAPPII and characterized its structure and subunit organization by single-particle EM. Our data show that the nine TRAPPII components form a core complex that dimerizes into a three-layered, diamond-shaped structure. The TRAPPI subunits assemble into TRAPPI complexes that form the outer layers. The three TRAPPII-specific subunits cap the ends of TRAPPI and form the middle layer, which is responsible for dimerization. TRAPPII binds the Ypt1 GTPase and probably uses the TRAPPI catalytic core to promote guanine nucleotide exchange. We discuss the implications of the structure of TRAPPII for coat interaction and TRAPPII-associated human pathologies.

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

NASA Astrophysics Data System (ADS)

Sciuto, Stephen V.; Liu, Jiangjiang; Konermann, Lars

2011-10-01

Electrosprayed multi-protein complexes can be dissociated by collisional activation in the gas phase. Typically, these processes follow a mechanism whereby a single subunit gets ejected with a disproportionately high amount of charge relative to its mass. This asymmetric behavior suggests that the departing subunit undergoes some degree of unfolding prior to being separated from the residual complex. These structural changes occur concomitantly with charge (proton) transfer towards the subunit that is being unraveled. Charge accumulation takes place up to the point where the subunit loses physical contact with the residual complex. This work develops a simple electrostatic model for studying the relationship between conformational changes and charge enrichment during collisional activation. Folded subunits are described as spheres that carry continuum surface charge. The unfolded chain is envisioned as random coil bead string. Simulations are guided by the principle that the system will adopt the charge configuration with the lowest potential energy for any backbone conformation. A finite-difference gradient algorithm is used to determine the charge on each subunit throughout the dissociation process. Both dimeric and tetrameric protein complexes are investigated. The model reproduces the occurrence of asymmetric charge partitioning for dissociation events that are preceded by subunit unfolding. Quantitative comparisons of experimental MS/MS data with model predictions yield estimates of the structural changes that occur during collisional activation. Our findings suggest that subunit separation can occur over a wide range of scission point structures that correspond to different degrees of unfolding.

Differences in the phenotypic effects of mutations in homologous MrpA and MrpD subunits of the multi-subunit Mrp-type Na+/H+ antiporter.

PubMed

Morino, Masato; Ogoda, Shinichiro; Krulwich, Terry Ann; Ito, Masahiro

2017-01-01

Mrp antiporters are the sole antiporters in the Cation/Proton Antiporter 3 family of transporter databases because of their unusual structural complexity, 6-7 hydrophobic proteins that function as a hetero-oligomeric complex. The two largest and homologous subunits, MrpA and MrpD, are essential for antiport activity and have direct roles in ion transport. They also show striking homology with proton-conducting, membrane-embedded Nuo subunits of respiratory chain complex I of bacteria, e.g., Escherichia coli. MrpA has the closest homology to the complex I NuoL subunit and MrpD has the closest homology to the complex I NuoM and N subunits. Here, introduction of mutations in MrpD, in residues that are also present in MrpA, led to defects in antiport function and/or complex formation. No significant phenotypes were detected in strains with mutations in corresponding residues of MrpA, but site-directed changes in the C-terminal region of MrpA had profound effects, showing that the MrpA C-terminal region has indispensable roles in antiport function. The results are consistent with a divergence in adaptations that support the roles of MrpA and MrpD in secondary antiport, as compared to later adaptations supporting homologs in primary proton pumping by the respiratory chain complex I.

Evolution of disorder in Mediator complex and its functional relevance.

PubMed

Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K

2016-02-29

Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of 'junction-MoRF' has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein-protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

PubMed

Ahmad-Sohdi, Nor-Ain-Shahajar; Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Hassan, Maizom

2015-01-01

Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.

Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves

PubMed Central

Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura

2015-01-01

Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with K m values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The K m values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. PMID:26600471

The Purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

PubMed Central

Derrien, Benoît; Majeran, Wojciech; Effantin, Grégory; Ebenezer, Joseph; Friso, Giulia; van Wijk, Klaas J.; Steven, Alasdair C.; Maurizi, Michael R.; Vallon, Olivier

2012-01-01

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits PMID:22772861

Clinicopathologic study of succinate-dehydrogenase-deficient gastrointestinal stromal tumors: A single-institutional experience in China.

PubMed

Liu, Weizhen; Zeng, Xiangyu; Wu, Xiuli; He, Jun; Gao, Jinbo; Shuai, Xiaoming; Wang, Guobin; Zhang, Peng; Tao, Kaixiong

2017-08-01

Gastrointestinal stromal tumors (GISTs) that are not driven by kinase mutations, as are most GISTs, often show loss of function of the succinate dehydrogenase (SDH) complex and are considered SDH-deficient GISTs. SDH-deficient GISTs share many distinct characteristics compared with conventional GISTs. However, data regarding these characteristics, particularly among Asian people, are relatively limited. The objective of this study was to characterize the clinicopathologic characteristics, treatment, and prognosis of these uncommon GISTs.This retrospective observational study enrolled 12 patients with SDH-deficient GISTs, who were selected from 335 patients with GIST diagnosed at our institution between October 31, 2013 and October 31, 2016 by succinate dehydrogenase subunit B staining.There were 8 male and 4 female patients, with a median age of 57 years (range, 21-73 years). Ten patients (83.3%) were diagnosed at or after the age of 40 years and represented 7.2% (10/138) of the entire population of elderly patients with gastric GISTs. The tumor size ranged from 3 to 19 cm (median, 7 cm); the primary tumor was multifocal in 6 cases (50%), and tumors had a multinodular or plexiform architecture in 10 cases (83.3%). Ten cases (83.3%) showed pure epithelioid morphology, with the remaining 2 cases (16.7%) showing mixed histologic subtype. Lymph node metastasis was found at the time of primary resection in 50% (3/6) of patients. Four cases (33.3%) had distant metastasis at presentation. Four patients (33.3%) developed disease progression during imatinib treatment after initial resection, but all of these patients regained disease control when the treatment was altered to sunitinib targeted therapy.SDH-deficient GISTs arise exclusively in the stomach and account for approximately 7.4% (12/162) of gastric GISTs. Moreover, those affecting people older than 40 years are not uncommon and sunitinib may work well for cases showing treatment failure with imatinib.

Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

ERIC Educational Resources Information Center

Strumilo, Slawomir

2005-01-01

Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex* ♦

PubMed Central

Nadeau, Owen W.; Lane, Laura A.; Xu, Dong; Sage, Jessica; Priddy, Timothy S.; Artigues, Antonio; Villar, Maria T.; Yang, Qing; Robinson, Carol V.; Zhang, Yang; Carlson, Gerald M.

2012-01-01

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)4 complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)2 lobes joined with D2 symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)4 complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)4 kinase core, because a β4 subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit. PMID:22969083

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

PubMed

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-05-02

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1-8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible "mini-complexes" or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetics Home Reference: pyruvate dehydrogenase deficiency

MedlinePlus

... form that cells can use. The pyruvate dehydrogenase complex converts a molecule called pyruvate, which is formed from the breakdown of carbohydrates, into another molecule called acetyl-CoA. This conversion ...

D-beta-hydroxybutyrate extends lifespan in C. elegans

PubMed Central

Edwards, Clare; Canfield, John; Copes, Neil; Rehan, Muhammad; Lipps, David; Bradshaw, Patrick C.

2014-01-01

The ketone body beta-hydroxybutyrate (βHB) is a histone deacetylase (HDAC) inhibitor and has been shown to be protective in many disease models, but its effects on aging are not well studied. Therefore we determined the effect of βHB supplementation on the lifespan of C. elegans nematodes. βHB supplementation extended mean lifespan by approximately 20%. RNAi knockdown of HDACs hda-2 or hda-3 also increased lifespan and further prevented βHB-mediated lifespan extension. βHB-mediated lifespan extension required the DAF-16/FOXO and SKN-1/Nrf longevity pathways, the sirtuin SIR-2.1, and the AMP kinase subunit AAK-2. βHB did not extend lifespan in a genetic model of dietary restriction indicating that βHB is likely functioning through a similar mechanism. βHB addition also upregulated βHB dehydrogenase activity and increased oxygen consumption in the worms. RNAi knockdown of F55E10.6, a short chain dehydrogenase and SKN-1 target gene, prevented the increased lifespan and βHB dehydrogenase activity induced by βHB addition, suggesting that F55E10.6 functions as an inducible βHB dehydrogenase. Furthermore, βHB supplementation increased worm thermotolerance and partially prevented glucose toxicity. It also delayed Alzheimer's amyloid-beta toxicity and decreased Parkinson's alpha-synuclein aggregation. The results indicate that D-βHB extends lifespan through inhibiting HDACs and through the activation of conserved stress response pathways. PMID:25127866

The α' subunit of β-conglycinin and the A1-5 subunits of glycinin are not essential for many hypolipidemic actions of dietary soy proteins in rats.

PubMed

Chen, Qixuan; Wood, Carla; Gagnon, Christine; Cober, Elroy R; Frégeau-Reid, Judith A; Gleddie, Stephen; Xiao, Chao Wu

2014-08-01

This study examined the effects of dietary soy protein (SP) lacking different storage protein subunits and isoflavones (ISF) on the abdominal fat, blood lipids, thyroid hormones, and enzymatic activities in rats. Weanling Sprague-Dawley rats (8 males and 8 females/group) were fed diets containing either 20 % casein without or with supplemental isoflavones or alcohol-washed SP isolate or SP concentrates (SPC) prepared from 6 different soy bean lines for 8 weeks. Feeding of diets containing SPC regardless of their subunit compositions significantly lowered relative liver weights, blood total, free, and LDL cholesterol in both genders (P < 0.05) and also reduced serum free fatty acids (FFA) and abdominal fat in females (P < 0.05) compared to the casein or casein + ISF diets. Dietary SPC significantly elevated the plasma free triiodothyronine (T3) in both genders and total T3 in females compared to the casein diet (P < 0.05). The SPC lacking β-conglycinin α' and either the glycinin A1-3 or A1-5 subunits increased total T3 in males and reduced plasma enzymatic activities of creatine kinase and lactate dehydrogenase compared to casein or casein + ISF diet (P < 0.05). Soy isoflavones were mainly responsible for the hypocholesterolemic effects and increased plasma free T3, whereas reduction in FFA, abdominal fat, liver weight and increased plasma total T3 were the effects of the soy proteins. Neither the α' subunit of β-conglycinin nor the A1-5 subunits of glycinin are essential for the hypolipidemic properties of soy proteins.

Whole Exome Sequencing Identifies the Genetic Basis of Late-Onset Leigh Syndrome in a Patient with MRI but Little Biochemical Evidence of a Mitochondrial Disorder.

PubMed

Nafisinia, Michael; Guo, Yiran; Dang, Xiao; Li, Jiankang; Chen, Yulan; Zhang, Jianguo; Lake, Nicole J; Gold, Wendy A; Riley, Lisa G; Thorburn, David R; Keating, Brendan; Xu, Xun; Hakonarson, Hakon; Christodoulou, John

2017-01-01

Leigh syndrome is a subacute necrotising encephalomyopathy proven by post-mortem analysis of brain tissue showing spongiform lesions with vacuolation of the neuropil followed by demyelination, gliosis and capillary proliferation caused by mutations in one of over 75 different genes, including nuclear- and mitochondrial-encoded genes, most of which are associated with mitochondrial respiratory chain function. In this study, we report a patient with suspected Leigh syndrome presenting with seizures, ptosis, scoliosis, dystonia, symmetrical putaminal abnormalities and a lactate peak on brain MRS, but showing normal MRC enzymology in muscle and liver, thereby complicating the diagnosis. Whole exome sequencing uncovered compound heterozygous mutations in NADH dehydrogenase (ubiquinone) flavoprotein 1 gene (NDUFV1), c.1162+4A>C (NM_007103.3), resulting in skipping of exon 8, and c.640G>A, causing the amino acid substitution p.Glu214Lys, both of which have previously been reported in a patient with complex I deficiency. Patient fibroblasts showed a significant reduction in NDUFV1 protein expression, decreased complex CI and complex IV assembly and consequential reductions in the enzymatic activities of both complexes by 38% and 67%, respectively. The pathogenic effect of these variations was further confirmed by immunoblot analysis of subunits for MRC enzyme complexes in patient muscle, liver and fibroblast where we observed 90%, 60% and 95% reduction in complex CI, respectively. Together these studies highlight the importance of a comprehensive, multipronged approach to the laboratory evaluation of patients with suspected Leigh syndrome.

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Genetics of bacteria that oxidize one-carbon compounds. Progress report, March 1, 1991--June 30, 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanson, R.S.

In the past several years researchers have identified at least 20 genes whose products were required for the oxidation of methanol to formaldehyde in three different facultative methylotrophic bacteria. These genes include structural genes for a cytochrome c{sub L} (mox G) and is a specific electron acceptor for methanol dehydrogenase (MDH), and the two structural genes that encode the large subunit (mox F) and smaller subunit (mox I) of MDH. Other genes are required for the synthesis of the prosthetic group of MDH, Pyrroloquinoline quinone (PQQ), and proteins required for assembly of the active MDH in the periplasm. Three genesmore » are believed to be required for incorporation of calcium into the MDH tetramer. The principal investigator`s group has studied the regulation of methanol oxidation in the pink-pigmented-facultative methylotroph Methylobacterium organophilum XX. The authors have mapped several genes and have sequenced the mox F gene and sequences upstream of mox F. The authors had tentatively identified several genes required for the transcription of the MDH structural genes in three methylotrophs. In the previous proposal, the P.I. proposed to establish an in-vitro transcription/translation system to study the function of the regulatory gene products. Further studies demonstrated that the regulation of transcription of these genes was far more complex than imagined at that time and the research plan was modified to determine the number and function of the regulatory genes using genetic approaches.« less

The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.

PubMed

Heath, Caroline; Posner, Mareike G; Aass, Hans C; Upadhyay, Abhishek; Scott, David J; Hough, David W; Danson, Michael J

2007-10-01

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

PubMed

Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

2003-05-01

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions

PubMed Central

Almeida, Daniela; Maldonado, Emanuel; Vasconcelos, Vitor; Antunes, Agostinho

2015-01-01

Mitochondrial protein-coding genes (mt genes) encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS). Despite the vital role of the mitochondrial genome (mt genome) in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments ranging from the intertidal to the deep sea, having distinct metabolic requirements, varied body shapes and highly advanced visual and nervous systems that make them highly competitive and successful worldwide predators. Thus, cephalopods are valuable models for testing natural selection acting on their mitochondrial subunits (mt subunits). Here, we used concatenated mt genes from 17 fully sequenced mt genomes of diverse cephalopod species to generate a robust mitochondrial phylogeny for the Class Cephalopoda. We followed an integrative approach considering several branches of interest–covering cephalopods with distinct morphologies, metabolic rates and habitats–to identify sites under positive selection and localize them in the respective protein alignment and/or tridimensional structure of the mt subunits. Our results revealed significant adaptive variation in several mt subunits involved in the energy production pathway of cephalopods: ND5 and ND6 from Complex I, CYTB from Complex III, COX2 and COX3 from Complex IV, and in ATP8 from Complex V. Furthermore, we identified relevant sites involved in protein-interactions, lining proton translocation channels, as well as disease/deficiencies related sites in the aforementioned complexes. A particular case, revealed by this study, is the involvement of some positively selected sites, found in Octopoda lineage in lining proton translocation channels (site 74 from ND5) and in interactions between subunits (site 507 from ND5) of Complex I. PMID:26285039

Assignment of function to Histidines 260 and 298 by engineering the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions that lead to acceptance of substrates lacking the 5-carboxyl group.†

PubMed Central

Shim, Da Jeong; Nemeria, Natalia S.; Balakrishnan, Anand; Patel, Hetalben; Song, Jaeyoung; Wang, Junjie; Jordan, Frank; Farinas, Edgardo T.

2011-01-01

The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced by hydrophobic residues of similar molecular volume. To interrogate whether the second component would enable synthesis of acyl-coenzymeA derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the ‘gatekeeper’ for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate, but E2o for 2-oxoglutarate. PMID:21809826

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury.

PubMed

Nowak, Grazyna; Takacsova-Bakajsova, Diana; Megyesi, Judit

2017-01-01

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia. Copyright © 2017 the American Physiological Society.

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury

PubMed Central

Takacsova-Bakajsova, Diana; Megyesi, Judit

2016-01-01

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia. PMID:27760765

Mitochondrial function in skeletal muscle of patients with protracted critical illness and ICU-acquired weakness.

PubMed

Jiroutková, Kateřina; Krajčová, Adéla; Ziak, Jakub; Fric, Michal; Waldauf, Petr; Džupa, Valér; Gojda, Jan; Němcova-Fürstová, Vlasta; Kovář, Jan; Elkalaf, Moustafa; Trnka, Jan; Duška, František

2015-12-24

Mitochondrial damage occurs in the acute phase of critical illness, followed by activation of mitochondrial biogenesis in survivors. It has been hypothesized that bioenergetics failure of skeletal muscle may contribute to the development of ICU-acquired weakness. The aim of the present study was to determine whether mitochondrial dysfunction persists until protracted phase of critical illness. In this single-centre controlled-cohort ex vivo proof-of-concept pilot study, we obtained vastus lateralis biopsies from ventilated patients with ICU-acquired weakness (n = 8) and from age and sex-matched metabolically healthy controls (n = 8). Mitochondrial functional indices were measured in cytosolic context by high-resolution respirometry in tissue homogenates, activities of respiratory complexes by spectrophotometry and individual functional capacities were correlated with concentrations of electron transport chain key subunits from respiratory complexes II, III, IV and V measured by western blot. The ability of aerobic ATP synthesis (OXPHOS) was reduced to ~54% in ICU patients (p<0.01), in correlation with the depletion of complexes III (~38% of control, p = 0.02) and IV (~26% of controls, p<0.01) and without signs of mitochondrial uncoupling. When mitochondrial functional indices were adjusted to citrate synthase activity, OXPHOS and the activity of complexes I and IV were not different, whilst the activities of complexes II and III were increased in ICU patients 3-fold (p<0.01) respectively 2-fold (p<0.01). Compared to healthy controls, in ICU patients we have demonstrated a ~50% reduction of the ability of skeletal muscle to synthetize ATP in mitochondria. We found a depletion of complex III and IV concentrations and relative increases in functional capacities of complex II and glycerol-3-phosphate dehydrogenase/complex III.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-09-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease

PubMed Central

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-01-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes. PMID:25333063

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

Regulation of Nuclear Import and Export of Negative Cofactor 2*S⃞

PubMed Central

Kahle, Joerg; Piaia, Elisa; Neimanis, Sonja; Meisterernst, Michael; Doenecke, Detlef

2009-01-01

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2α and NC2β, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2α and NC2β depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin α/β heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2β. In contrast, NC2α does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2α masks the NES in NC2β, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin α/β-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. PMID:19204005

Molecular architecture of the yeast Mediator complex

PubMed Central

Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo; Greenberg, Charles H; Bushnell, David A; Davis, Ralph; Burlingame, Alma L; Sali, Andrej; Kornberg, Roger D

2015-01-01

The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex. DOI: http://dx.doi.org/10.7554/eLife.08719.001 PMID:26402457

Electron transfer flavoprotein deficiency: functional and molecular aspects.

PubMed

Schiff, Manuel; Froissart, Roseline; Olsen, Rikke K J; Acquaviva, Cécile; Vianey-Saban, Christine

2006-06-01

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a recessively inherited metabolic disorder that can be due to a deficiency of electron transfer flavoprotein (ETF) or its dehydrogenase (ETF-ubiquinone oxidoreductase). ETF is a mitochondrial matrix protein consisting of alpha- (30kDa) and beta- (28kDa) subunits encoded by the ETFA and ETFB genes, respectively. In the present study, we have analysed tissue samples from 16 unrelated patients with ETF deficiency, and we report the results of ETF activity, Western blot analysis and mutation analysis. The ETF assay provides a reliable diagnostic tool to confirm ETF deficiency in patients suspected to suffer from MADD. Activity ranged from less than 1 to 16% of controls with the most severely affected patients disclosing the lowest activity values. The majority of patients had mutations in the ETFA gene while only two of them harboured mutations in the ETFB gene. Nine novel disease-causing ETF mutations are reported.

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

NASA Astrophysics Data System (ADS)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

PubMed

Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

2011-06-01

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

3D structure of the influenza virus polymerase complex: Localization of subunit domains

PubMed Central

Area, Estela; Martín-Benito, Jaime; Gastaminza, Pablo; Torreira, Eva; Valpuesta, José M.; Carrascosa, José L.; Ortín, Juan

2004-01-01

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP–mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP. PMID:14691253

Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes.

PubMed

Hassan, Ahmed H; Prochasson, Philippe; Neely, Kristen E; Galasinski, Scott C; Chandy, Mark; Carrozza, Michael J; Workman, Jerry L

2002-11-01

The functions of the SAGA and SWI/SNF complexes are interrelated and can form stable "epigenetic marks" on promoters in vivo. Here we show that stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation. This acetylation can be brought about by either the SAGA or NuA4 HAT complexes. The bromodomain in the Spt7 subunit of SAGA is dispensable for this activity but will anchor SAGA if it is swapped into Gcn5, indicating that specificity of bromodomain function is determined in part by the subunit it occupies. Thus, bromodomains within the catalytic subunits of SAGA and SWI/SNF anchor these complexes to acetylated promoter nucleosomes.

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

PubMed Central

Tramonti, Angela; Lanini, Claudio; Cialfi, Samantha; De Biase, Daniela; Falcone, Claudio

2012-01-01

In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes. PMID:23064253

Methylation-regulated decommissioning of multimeric PP2A complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li

2017-12-01

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivationmore » of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.« less

Highly acidic C-terminal domain of pp32 is required for the interaction with histone chaperone, TAF-Ibeta.

PubMed

Lee, In-Seon; Oh, Sang-Min; Kim, Sung-Mi; Lee, Dong-Seok; Seo, Sang-Beom

2006-12-01

We have previously reported that INHAT (inhibitor of acetyltransferases) complex subunits, TAF (template activating factor)-Ialpha, TAF-Ibeta and pp32 can inhibit histone acetylation and HAT (histone acetyltransferase)-dependent transcription by binding to histones. Evidences are accumulating that INHAT complex subunits have important regulatory roles in various cellular activities such as replication, transcription, and apoptosis etc. However, how these subunits interact each other remains largely unknown. Using immunoprecipitation (IP) and protein-protein interaction assays with TAF-Ibeta and pp32 deletion mutant proteins, we identify INHAT complex subunits, TAF-Ibeta and pp32 interaction requires highly acidic C-terminal domain of pp32. We also show that the interaction between the INHAT complex subunits is stronger in the presence of histones. In this study, we report that the synergistic inhibition of HAT-mediated transcription by TAF-Ibeta and pp32 is dependent on the highly acidic C-terminal domain of pp32.

Effect of chlorocamphene on the isoenzyme spectrum of lactate dehydrogenase in rat serum and liver.

PubMed Central

Kuz'minskaya, U A; Alekhina, S M

1976-01-01

Rats were used to study the general activity and the isoenzyme spectrum of lactate dehydrogenase (LDH) during single-instance and long-term introduction of polychlorocamphene. Total lactate dehydrogenase activity decreases in the liver during the single-instance introduction of half the LD50 (120 mg/kg). The isoenzyme spectrum of LDH is characterized by an increase in the quantity of LDH1, LDH2, and LDH3 and by a decrease in the amount of LDH4. The overall LDH activity does not change in blood serum. The isoform ratio changes insignificantly and LDH1 falls, but normalized 15 days after the introduction of the compound. Long-term introduction of polychlorocamphene at levels 1/100 the LD50 dose over 1.3 and 6 months causes a reduction in the overall LDH activity, both in the liver and in the serum. A decrease in the activity of the basic LDH isoenzyme of the liver (LDH5) and a sharp increase in LDH3 are characteristic for the isoenzyme spectrum of the liver. LDH1 and LDH4 decrease and LDH2 and LDH3 increase in blood serum. Beginning with the third month of polychlorocamphene introduction, LDH1 tends to return to normal levels. LDH2, LDH3, and LDH4 do return to normal levels, while LDH5 increases regularly. This results in a reduction of the number of H subunits and an increase of M subunits. This is characteristic of hypoxic states. On comparing the changes in the LDH enzymes of the liver and blood serum, it can be considered that the introduction of polychlorocamphene does not result in an increase in the permeability of the cellular membranes of the liver for LDH isoenzymes, while the observed isoenzyme spectrum shifts in blood serum are either the result of the biosynthesis of the isoforms of this enzyme changed by the compound or the result of the permeability for them of cells of other tissues. PMID:1269500

Electrochemical, photoelectrochemical, and surface plasmon resonance detection of cocaine using supramolecular aptamer complexes and metallic or semiconductor nanoparticles.

PubMed

Golub, Eyal; Pelossof, Gilad; Freeman, Ronit; Zhang, Hong; Willner, Itamar

2009-11-15

Metallic or semiconductor nanoparticles (NPs) are used as labels for the electrochemical, photoelectrochemical, or surface plasmon resonance (SPR) detection of cocaine using a common aptasensor configuration. The aptasensors are based on the use of two anticocaine aptamer subunits, where one subunit is assembled on a Au support, acting as an electrode or a SPR-active surface, and the second aptamer subunit is labeled with Pt-NPs, CdS-NPs, or Au-NPs. In the different aptasensor configurations, the addition of cocaine results in the formation of supramolecular complexes between the NPs-labeled aptamer subunits and cocaine on the metallic surface, allowing the quantitative analysis of cocaine. The supramolecular Pt-NPs-aptamer subunits-cocaine complex allows the detection of cocaine by the electrocatalyzed reduction of H(2)O(2). The photocurrents generated by the CdS-NPs-labeled aptamer subunits-cocaine complex, in the presence of triethanol amine as a hole scavenger, allows the photoelectrochemical detection of cocaine. The supramolecular Au-NPs-aptamer subunits-cocaine complex generated on the Au support allows the SPR detection of cocaine through the reflectance changes stimulated by the electronic coupling between the localized plasmon of the Au-NPs and the surface plasmon wave. All aptasensor configurations enable the analysis of cocaine with a detection limit in the range of 10(-6) to 10(-5) M. The major advantage of the sensing platform is the lack of background interfering signals.

Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

PubMed

Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

2016-12-01

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

PubMed

Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

2013-05-01

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

PubMed Central

2013-01-01

Background Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Results Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. Conclusions SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights. PMID:23635033

Discovery of a potent and highly specific β2 proteasome inhibitor from a library of copper complexes.

PubMed

Zhou, Tongliang; Cai, Yuanbo; Liang, Lei; Yang, Lingfei; Xu, Fengrong; Niu, Yan; Wang, Chao; Zhang, Jun-Long; Xu, Ping

2016-12-01

We reported the synthesis, characterization and biological activity of several copper(II) Schiff base complexes, which exhibit high proteasome inhibitory activities with particular selectivity of β 2 subunit. Structure-activity relationships information obtained from complex Na 2 [Cu(a4s1)] demonstrated that distinct bonding modes in β 2 and β 5 subunits determines its selectivity and potent inhibition for β 2 subunit. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioenergetics of Stromal Cells as a Predictor of Aggressive Prostate Cancer

DTIC Science & Technology

2016-11-01

complex tissue preparations (human prostate and prostatic adenoma) and rat ventral prostate cells it was reported to exhibit high aerobic glycolysis [19...pyruvate dehydrogenase kinase), 2DG (inhibitor of hexokinase), or metformin (inhibitor of mitochondrial complex I) [41] as a therapeutic approach to... cyanide 4-(trifluoromethoxy) phenylhydrazone; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GlyST, Glycolytic stress test; HPV, human papilloma virus

Genetic expansion of chaperonin-containing TCP-1 (CCT/TRiC) complex subunits yields testis-specific isoforms required for spermatogenesis in planarian flatworms.

PubMed

Counts, Jenna T; Hester, Tasha M; Rouhana, Labib

2017-12-01

Chaperonin-containing Tail-less complex polypeptide 1 (CCT) is a highly conserved, hetero-oligomeric complex that ensures proper folding of actin, tubulin, and regulators of mitosis. Eight subunits (CCT1-8) make up this complex, and every subunit has a homolog expressed in the testes and somatic tissue of the planarian flatworm Schmidtea mediterranea. Gene duplications of four subunits in the genomes of S. mediterranea and other planarian flatworms created paralogs to CCT1, CCT3, CCT4, and CCT8 that are expressed exclusively in the testes. Functional analyses revealed that each CCT subunit expressed in the S. mediterranea soma is essential for homeostatic integrity and survival, whereas sperm elongation defects were observed upon knockdown of each individual testis-specific paralog (Smed-cct1B; Smed-cct3B; Smed-cct4A; and Smed-cct8B), regardless of potential redundancy with paralogs expressed in both testes and soma (Smed-cct1A; Smed-cct3A; Smed-cct4B; and Smed-cct8A). Yet, no detriment was observed in the number of adult somatic stem cells (neoblasts) that maintain differentiated tissue in planarians. Thus, expression of all eight CCT subunits is required to execute the essential functions of the CCT complex. Furthermore, expression of the somatic paralogs in planarian testes is not sufficient to complete spermatogenesis when testis-specific paralogs are knocked down, suggesting that the evolution of chaperonin subunits may drive changes in the development of sperm structure and that correct CCT subunit stoichiometry is crucial for spermiogenesis. © 2017 Wiley Periodicals, Inc.

Levels of control exerted by the Isc iron-sulfur cluster system on biosynthesis of the formate hydrogenlyase complex.

PubMed

Pinske, Constanze; Jaroschinsky, Monique; Sawers, R Gary

2013-06-01

The membrane-associated formate hydrogenlyase (FHL) complex of bacteria like Escherichia coli is responsible for the disproportionation of formic acid into the gaseous products carbon dioxide and dihydrogen. It comprises minimally seven proteins including FdhF and HycE, the catalytic subunits of formate dehydrogenase H and hydrogenase 3, respectively. Four proteins of the FHL complex have iron-sulphur cluster ([Fe-S]) cofactors. Biosynthesis of [Fe-S] is principally catalysed by the Isc or Suf systems and each comprises proteins for assembly and for delivery of [Fe-S]. This study demonstrates that the Isc system is essential for biosynthesis of an active FHL complex. In the absence of the IscU assembly protein no hydrogen production or activity of FHL subcomponents was detected. A deletion of the iscU gene also resulted in reduced intracellular formate levels partially due to impaired synthesis of pyruvate formate-lyase, which is dependent on the [Fe-S]-containing regulator FNR. This caused reduced expression of the formate-inducible fdhF gene. The A-type carrier (ATC) proteins IscA and ErpA probably deliver [Fe-S] to specific apoprotein components of the FHL complex because mutants lacking either protein exhibited strongly reduced hydrogen production. Neither ATC protein could compensate for the lack of the other, suggesting that they had independent roles in [Fe-S] delivery to complex components. Together, the data indicate that the Isc system modulates FHL complex biosynthesis directly by provision of [Fe-S] as well as indirectly by influencing gene expression through the delivery of [Fe-S] to key regulators and enzymes that ultimately control the generation and oxidation of formate.

Concomitant loss of NDH complex-related genes within chloroplast and nuclear genomes in some orchids.

PubMed

Lin, Choun-Sea; Chen, Jeremy J W; Chiu, Chi-Chou; Hsiao, Han C W; Yang, Chen-Jui; Jin, Xiao-Hua; Leebens-Mack, James; de Pamphilis, Claude W; Huang, Yao-Ting; Yang, Ling-Hung; Chang, Wan-Jung; Kui, Ling; Wong, Gane Ka-Shu; Hu, Jer-Ming; Wang, Wen; Shih, Ming-Che

2017-06-01

The chloroplast NAD(P)H dehydrogenase-like (NDH) complex consists of about 30 subunits from both the nuclear and chloroplast genomes and is ubiquitous across most land plants. In some orchids, such as Phalaenopsis equestris, Dendrobium officinale and Dendrobium catenatum, most of the 11 chloroplast genome-encoded ndh genes (cp-ndh) have been lost. Here we investigated whether functional cp-ndh genes have been completely lost in these orchids or whether they have been transferred and retained in the nuclear genome. Further, we assessed whether both cp-ndh genes and nucleus-encoded NDH-related genes can be lost, resulting in the absence of the NDH complex. Comparative analyses of the genome of Apostasia odorata, an orchid species with a complete complement of cp-ndh genes which represents the sister lineage to all other orchids, and three published orchid genome sequences for P. equestris, D. officinale and D. catenatum, which are all missing cp-ndh genes, indicated that copies of cp-ndh genes are not present in any of these four nuclear genomes. This observation suggests that the NDH complex is not necessary for some plants. Comparative genomic/transcriptomic analyses of currently available plastid genome sequences and nuclear transcriptome data showed that 47 out of 660 photoautotrophic plants and all the heterotrophic plants are missing plastid-encoded cp-ndh genes and exhibit no evidence for maintenance of a functional NDH complex. Our data indicate that the NDH complex can be lost in photoautotrophic plant species. Further, the loss of the NDH complex may increase the probability of transition from a photoautotrophic to a heterotrophic life history. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Activity, polypeptide and gene identification of thylakoid Ndh complex in trees: potential physiological relevance of fluorescence assays.

PubMed

Serrot, Patricia H; Sabater, Bartolomé; Martín, Mercedes

2012-09-01

Three evergreen (Laurus nobilis, Viburnum tinus and Thuja plicata) and two autumnal abscission deciduous trees (Cydonia oblonga and Prunus domestica) have been investigated for the presence (zymogram and immunodetection) and functionality (post-illumination chlorophyll fluorescence) of the thylakoid Ndh complex. The presence of encoding ndh genes has also been investigated in T. plicata. Western assays allowed tentative identification of zymogram NADH dehydrogenase bands corresponding to the Ndh complex after native electrophoresis of solubilized fractions from L. nobilis, V. tinus, C. oblonga and P. domestica leaves, but not in those of T. plicata. However, Ndh subunits were detected after SDS-PAGE of thylakoid solubilized proteins of T. plicata. The leaves of the five plants showed the post-illumination chlorophyll fluorescence increase dependent on the presence of active Ndh complex. The fluorescence increase was higher in autumn in deciduous, but not in evergreen trees, which suggests that the thylakoid Ndh complex could be involved in autumnal leaf senescence. Two ndhB genes were sequenced from T. plicata that differ at the 350 bp 3' end sequence. Comparison with the mRNA revealed that ndhB genes have a 707-bp type II intron between exons 1 (723 bp) and 2 (729 bp) and that the UCA 259th codon is edited to UUA in mRNA. Phylogenetically, the ndhB genes of T. plicata group close to those of Metasequoia, Cryptomeria, Taxodium, Juniperus and Widdringtonia in the cupresaceae branch and are 5' end shortened by 18 codons with respect to that of angiosperms. Copyright © Physiologia Plantarum 2012.

Activity, polypeptide and gene identification of thylakoid Ndh complex in trees: potential physiological relevance of fluorescence assays

PubMed Central

Serrot, Patricia H; Sabater, Bartolomé; Martín, Mercedes

2012-01-01

Three evergreen (Laurus nobilis, Viburnum tinus and Thuja plicata) and two autumnal abscission deciduous trees (Cydonia oblonga and Prunus domestica) have been investigated for the presence (zymogram and immunodetection) and functionality (post-illumination chlorophyll fluorescence) of the thylakoid Ndh complex. The presence of encoding ndh genes has also been investigated in T. plicata. Western assays allowed tentative identification of zymogram NADH dehydrogenase bands corresponding to the Ndh complex after native electrophoresis of solubilized fractions from L. nobilis, V. tinus, C. oblonga and P. domestica leaves, but not in those of T. plicata. However, Ndh subunits were detected after SDS-PAGE of thylakoid solubilized proteins of T. plicata. The leaves of the five plants showed the post-illumination chlorophyll fluorescence increase dependent on the presence of active Ndh complex. The fluorescence increase was higher in autumn in deciduous, but not in evergreen trees, which suggests that the thylakoid Ndh complex could be involved in autumnal leaf senescence. Two ndhB genes were sequenced from T. plicata that differ at the 350 bp 3′ end sequence. Comparison with the mRNA revealed that ndhB genes have a 707-bp type II intron between exons 1 (723 bp) and 2 (729 bp) and that the UCA 259th codon is edited to UUA in mRNA. Phylogenetically, the ndhB genes of T. plicata group close to those of Metasequoia, Cryptomeria, Taxodium, Juniperus and Widdringtonia in the cupresaceae branch and are 5′ end shortened by 18 codons with respect to that of angiosperms. PMID:22324908

Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

PubMed

Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J; Blombach, Bastian

2013-09-01

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

PubMed Central

Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J.

2013-01-01

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products. PMID:23835179

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

PubMed

Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

2006-06-02

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Non-functional plastid ndh gene fragments are present in the nuclear genome of Norway spruce (Picea abies L. Karsch): insights from in silico analysis of nuclear and organellar genomes.

PubMed

Ranade, Sonali Sachin; García-Gil, María Rosario; Rosselló, Josep A

2016-04-01

Many genes have been lost from the prokaryote plastidial genome during the early events of endosymbiosis in eukaryotes. Some of them were definitively lost, but others were relocated and functionally integrated to the host nuclear genomes through serial events of gene transfer during plant evolution. In gymnosperms, plastid genome sequencing has revealed the loss of ndh genes from several species of Gnetales and Pinaceae, including Norway spruce (Picea abies). This study aims to trace the ndh genes in the nuclear and organellar Norway spruce genomes. The plastid genomes of higher plants contain 11 ndh genes which are homologues of mitochondrial genes encoding subunits of the proton-pumping NADH-dehydrogenase (nicotinamide adenine dinucleotide dehydrogenase) or complex I (electron transport chain). Ndh genes encode 11 NDH polypeptides forming the Ndh complex (analogous to complex I) which seems to be primarily involved in chloro-respiration processes. We considered ndh genes from the plastidial genome of four gymnosperms (Cryptomeria japonica, Cycas revoluta, Ginkgo biloba, Podocarpus totara) and a single angiosperm species (Arabidopsis thaliana) to trace putative homologs in the nuclear and organellar Norway spruce genomes using tBLASTn to assess the evolutionary fate of ndh genes in Norway spruce and to address their genomic location(s), structure, integrity and functionality. The results obtained from tBLASTn were subsequently analyzed by performing homology search for finding ndh specific conserved domains using conserved domain search. We report the presence of non-functional plastid ndh gene fragments, excepting ndhE and ndhG genes, in the nuclear genome of Norway spruce. Regulatory transcriptional elements like promoters, TATA boxes and enhancers were detected in the upstream regions of some ndh fragments. We also found transposable elements in the flanking regions of few ndh fragments suggesting nuclear rearrangements in those regions. These evidences support the hypothesis that, at least in Picea, ndh translocations from the plastid to the nuclear genome have occurred, and that there might have been a functional machinery at some time during evolution to accommodate them within a nuclear-encoded environment, or attempts to form it.

High prevalence of carriers of variant c.1528G>C of HADHA gene causing long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the population of adult Kashubians from North Poland

PubMed Central

Nedoszytko, Bogusław; Siemińska, Alicja; Dąbrowski, Sławomir; Słomka, Marcin; Sobalska-Kwapis, Marta; Marciniak, Błażej; Wierzba, Jolanta; Skokowski, Jarosław; Fijałkowski, Marcin; Nowicki, Roman; Kalinowski, Leszek

2017-01-01

Background/Objectives The mitochondrial β-oxidation of fatty acids is a complex catabolic pathway. One of the enzymes of this pathway is the heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits. Mutations in MTP genes (HADHA and HADHB), both located on chromosome 2p23, cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by decreased activity of MTP. The most common MTP mutation is long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency caused by the c.1528G>C (rs137852769, p.Glu510Gln) substitution in exon 15 of the HADHA gene. Subjects/Methods We analyzed the frequency of genetic variants in the HADHA gene in the adults of Kashubian origin from North Poland and compared this data in other Polish provinces. Results We found a significantly higher frequency of HDHA c.1528G>C (rs137852769, p.Glu510Gln) carriers among Kashubians (1/57) compared to subjects from other regions of Poland (1/187). We found higher frequency of c.652G>C (rs71441018, pVal218Leu) polymorphism in the HADHA gene within population of Silesia, southern Poland (1/107) compared to other regions. Conclusion Our study indicate described high frequency of c.1528G>C variant of HADHA gene in Kashubian population, suggesting the founder effect. For the first time we have found high frequency of rs71441018 in the South Poland Silesian population. PMID:29095929

Peripheral stator of the yeast V-ATPase: stoichiometry and specificity of interaction between the EG complex and subunits C and H.

PubMed

Féthière, James; Venzke, David; Madden, Dean R; Böttcher, Bettina

2005-12-06

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.

Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy.

PubMed

Masai, J; Shibata, T; Kagawa, Y; Kondo, S

1992-07-01

Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium. The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG). The F1 subunit complex of the H(+)-ATPase, and an H(+)-ATPase whose F0 portion was embedded into liposomes prepared from soybean lecithin were imaged. Overall structural images of the subunit complex F1 were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation. Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX.

PKA modulation of Kv4.2-encoded A-type potassium channels requires formation of a supramolecular complex.

PubMed

Schrader, Laura A; Anderson, Anne E; Mayne, Amber; Pfaffinger, Paul J; Sweatt, John David

2002-12-01

A-type channels, encoded by the pore-forming alpha-subunits of the Kv4.x family, are particularly important in regulating membrane excitability in the CNS and the heart. Given the key role of modulation of A currents by kinases, we sought to investigate the protein structure-function relationships underlying the regulation of these currents by PKA. We have previously shown the existence of two PKA phosphorylation sites in the Kv4.2 sequence; therefore, we focused this study on the Kv4.2 primary subunit. In the present studies we made the surprising finding that PKA phosphorylation of the Kv4.2 alpha-subunit is necessary but not sufficient for channel modulation; channel modulation by PKA required the presence of an ancillary subunit, the K+ channel interacting protein (KChIP3). Therefore, these findings indicate a surprising complexity to kinase regulation of A currents, in that an interaction of two separate molecular events, alpha-subunit phosphorylation and the association of an ancillary subunit (KChIP3), are necessary for phosphorylation-dependent regulation of Kv4.2-encoded A channels by PKA. Overall, our studies indicate that PKA must of necessity act on a supramolecular complex of pore-forming alpha-subunits plus ancillary subunits to alter channel properties.

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.

PubMed

Ismaya, Wangsa T; Rozeboom, Henriëtte J; Weijn, Amrah; Mes, Jurriaan J; Fusetti, Fabrizia; Wichers, Harry J; Dijkstra, Bauke W

2011-06-21

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

Functional Differentiation of SWI/SNF Remodelers in Transcription and Cell Cycle Control▿ †

PubMed Central

Moshkin, Yuri M.; Mohrmann, Lisette; van Ijcken, Wilfred F. J.; Verrijzer, C. Peter

2007-01-01

Drosophila BAP and PBAP represent two evolutionarily conserved subclasses of SWI/SNF chromatin remodelers. The two complexes share the same core subunits, including the BRM ATPase, but differ in a few signature subunits: OSA defines BAP, whereas Polybromo (PB) and BAP170 specify PBAP. Here, we present a comprehensive structure-function analysis of BAP and PBAP. An RNA interference knockdown survey revealed that the core subunits BRM and MOR are critical for the structural integrity of both complexes. Whole-genome expression profiling suggested that the SWI/SNF core complex is largely dysfunctional in cells. Regulation of the majority of target genes required the signature subunit OSA, PB, or BAP170, suggesting that SWI/SNF remodelers function mostly as holoenzymes. BAP and PBAP execute similar, independent, or antagonistic functions in transcription control and appear to direct mostly distinct biological processes. BAP, but not PBAP, is required for cell cycle progression through mitosis. Because in yeast the PBAP-homologous complex, RSC, controls cell cycle progression, our finding reveals a functional switch during evolution. BAP mediates G2/M transition through direct regulation of string/cdc25. Its signature subunit, OSA, is required for directing BAP to the string/cdc25 promoter. Our results suggest that the core subunits play architectural and enzymatic roles but that the signature subunits determine most of the functional specificity of SWI/SNF holoenzymes in general gene control. PMID:17101803

The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

PubMed

Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

2011-01-01

The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans.

PubMed

Svetlitchnyi, V; Peschel, C; Acker, G; Meyer, O

2001-09-01

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H(2)O --> CO(2) + 2 e(-) + 2 H(+). Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) micromol of CO oxidized min(-1) mg(-1) of protein (methyl viologen, pH 8.0, 70 degrees C). The two enzymes oxidize CO very efficiently, as indicated by k(cat)/K(m) values at 70 degrees C of 1.3. 10(9) M(-1) CO s(-1) (CODH I) and 1.7. 10(9) M(-1) CO s(-1) (CODH II). The apparent K(m) values at pH 8.0 and 70 degrees C are 30 and 18 microM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.

Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

PubMed

Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

2010-03-23

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

Two Membrane-Associated NiFeS-Carbon Monoxide Dehydrogenases from the Anaerobic Carbon-Monoxide-Utilizing Eubacterium Carboxydothermus hydrogenoformans

PubMed Central

Svetlitchnyi, Vitali; Peschel, Christine; Acker, Georg; Meyer, Ortwin

2001-01-01

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H2O → CO2 + 2 e− + 2 H+. Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) μmol of CO oxidized min−1 mg−1 of protein (methyl viologen, pH 8.0, 70°C). The two enzymes oxidize CO very efficiently, as indicated by kcat/Km values at 70°C of 1.3 · 109 M−1 CO s−1 (CODH I) and 1.7 · 109 M−1 CO s−1 (CODH II). The apparent Km values at pH 8.0 and 70°C are 30 and 18 μM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions. PMID:11489867

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Distribution of anaerobic carbon monoxide dehydrogenase genes in deep subseafloor sediments.

PubMed

Hoshino, T; Inagaki, F

2017-05-01

Carbon monoxide (CO) is the simplest oxocarbon generated by the decomposition of organic compounds, and it is expected to be in marine sediments in substantial amounts. However, the availability of CO in the deep subseafloor sedimentary biosphere is largely unknown even though anaerobic oxidation of CO is a thermodynamically favourable reaction that possibly occurs with sulphate reduction, methanogenesis, acetogenesis and hydrogenesis. In this study, we surveyed for the first time the distribution of the CO dehydrogenase gene (cooS), which encodes the catalytic beta subunit of anaerobic CO dehydrogenase (CODH), in subseafloor sediment-core samples from the eastern flank of the Juan de Fuca Ridge, Mars-Ursa Basin, Kumano Basin, and off the Shimokita Peninsula, Japan, during Integrated Ocean Drilling Program (IODP) Expeditions 301, 308 and 315 and the D/V Chikyu shakedown cruise CK06-06, respectively. Our results show the occurrence of diverse cooS genes from the seafloor down to about 390 m below the seafloor, suggesting that microbial communities have metabolic functions to utilize CO in anoxic microbial ecosystems beneath the ocean floor, and that the microbial community potentially responsible for anaerobic CO oxidation differs in accordance with possible energy-yielding metabolic reactions in the deep subseafloor sedimentary biosphere. Little is known about the microbial community associated with carbon monoxide (CO) in the deep subseafloor. This study is the first survey of a functional gene encoding anaerobic carbon monoxide dehydrogenase (CODH). The widespread occurrence of previously undiscovered CO dehydrogenase genes (cooS) suggests that diverse micro-organisms are capable of anaerobic oxidation of CO in the deep subseafloor sedimentary biosphere. © 2017 The Society for Applied Microbiology.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms

PubMed Central

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-01-01

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. PMID:27044515

The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae

PubMed Central

Chen, Zhiqiang; Speck, Christian; Wendel, Patricia; Tang, Chunyan; Stillman, Bruce; Li, Huilin

2008-01-01

The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC–Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1–5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit–subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1–DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC. PMID:18647841

Multilocus sequence analysis of Echinococcus granulosus strains isolated from humans and animals in Iran.

PubMed

Nikmanesh, Bahram; Mirhendi, Hossein; Mahmoudi, Shahram; Rokni, Mohammad Bagher

2017-12-01

Echinococcus granulosus is now considered a complex consisting of at least four species and ten genotypes. Different molecular targets have been described for molecular characterization of E. granulosus; however, in almost all studies only one or two of the targets have been used, and only limited data is available on the utilization of multiple loci. Therefore, we investigated the genetic diversity among 64 strains isolated from 138 cyst specimens of human and animal isolates, using a set of nuclear and mitochondrial genes; i.e., cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), ATPase subunit 6 (atp6), 12S rRNA (12S), and Actin II (act II). In comparison to the use of molecular reference targets (nad1 + cox1), using singular target (act II or 12S or atp6) yielded lower discriminatory power. Act II and 12S genes could accurately discriminate the G6 genotype, but they were not able to differentiate between G1 and G3 genotypes. As the G1 and G3 genotypes belong to the E. granulosus sensu stricto, low intra-species variation was observed for act II and 12S. The atp6 gene could identify the G3 genotype but could not differentiate G6 and G1 genotypes. Using concatenated sequence of five genes (cox1 + nad1 + atp6 + 12S + act II), genotypes were identified accurately, and markedly higher resolution was obtained in comparison with the use of reference markers (nad1 + cox1) only. Application of multilocus sequence analysis (MLSA) to large-scale studies could provide valuable epidemiological data to make efficient control and management measures for cystic echinococcosis. Copyright © 2017 Elsevier Inc. All rights reserved.

MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype

PubMed Central

Hong, Zhigang; Chen, Kuang-Hueih; DasGupta, Asish; Potus, Francois; Dunham-Snary, Kimberly; Bonnet, Sebastien; Tian, Lian; Fu, Jennifer; Breuils-Bonnet, Sandra; Provencher, Steeve; Wu, Danchen; Mewburn, Jeffrey; Ormiston, Mark L.

2017-01-01

Rationale: Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer-like phenotype is promoted by increased cytosolic calcium ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH’s cancer-like phenotype. Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore-forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid-mediated up-regulation, as well as through modulation of the upstream microRNAs (miRs) miR-138 and miR-25. In vivo, nebulized anti-miRs were administered to rats with monocrotaline-induced PAH. Measurements and Main Results: Impaired MCUC function, resulting from down-regulation of MCU and up-regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAH’s pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR-138) down-regulate MCU directly and also by decreasing MCU’s transcriptional regulator cAMP response element–binding protein 1. Nebulized anti-miRs against miR-25 and miR-138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model. Conclusions: These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted. PMID:27648837

Structural Model for the Flip-Flop Action in Thiamin Pyrophosphate-Dependent Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2003-01-01

The derivative of vitamin B1 thiamin pyrophosphate (TPP) is a cofactor of enzymes performing catalysis in pathways of energy production, including (i) decarboxylation of alpha-keto acids followed by (ii) transketolation. These enzymes have shown a common mechanism of TPP activation by imposing an active V-conformation of this coenzyme that brings the N4 atom of the aminopyrimidine ring to the distance required for the intramolecular C-H N hydrogen-bonding with the C2- atom of the thiazolium ring. The reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used by the TPP-dependent enzymes. The structure of the heterotetrameric human pyruvate dehydrogenase (Elp) recently determined in our laboratory (1) revealed the association pattern of the subunits and the specifics of two chemically equivalent cofactor binding sites. Dynamic nonequivalence of these two cofactor sites directs the flip-flop action of this enzyme, depending upon which two active sites effect each other (2). The crystal structure derived from the holo-form of Elp provided the basis for the model of the flip-flop action of Elp in which different steps of the catalytic reaction are performed in each of the two cofactor sites at any given moment, where these steps are governed by the concerted shuttle-like motion of the subunits. It is further proposed that balancing a hydrogen-bond network and related cofactor geometry determine the continuity of catalytic events.

Embryonic transcriptome and proteome analyses on hepatic lipid metabolism in chickens divergently selected for abdominal fat content.

PubMed

Na, Wei; Wu, Yuan-Yuan; Gong, Peng-Fei; Wu, Chun-Yan; Cheng, Bo-Han; Wang, Yu-Xiang; Wang, Ning; Du, Zhi-Qiang; Li, Hui

2018-05-23

In avian species, liver is the main site of de novo lipogenesis, and hepatic lipid metabolism relates closely to adipose fat deposition. Using our fat and lean chicken lines of striking differences in abdominal fat content, post-hatch lipid metabolism in both liver and adipose tissues has been studied extensively. However, whether molecular discrepancy for hepatic lipid metabolism exists in chicken embryos remains obscure. We performed transcriptome and proteome profiling on chicken livers at five embryonic stages (E7, E12, E14, E17 and E21) between the fat and lean chicken lines. At each stage, 521, 141, 882, 979 and 169 differentially expressed genes were found by the digital gene expression, respectively, which were significantly enriched in the metabolic, PPAR signaling and fatty acid metabolism pathways. Quantitative proteomics analysis found 20 differentially expressed proteins related to lipid metabolism, PPAR signaling, fat digestion and absorption, and oxidative phosphorylation pathways. Combined analysis showed that genes and proteins related to lipid transport (intestinal fatty acid-binding protein, nucleoside diphosphate kinase, and apolipoprotein A-I), lipid clearance (heat shock protein beta-1) and energy metabolism (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and succinate dehydrogenase flavoprotein subunit) were significantly differentially expressed between the two lines. For hepatic lipid metabolism at embryonic stages, molecular differences related to lipid transport, lipid clearance and energy metabolism exist between the fat and lean chicken lines, which might contribute to the striking differences of abdominal fat deposition at post-hatch stages.

Molecular study of electron transfer flavoprotein alpha-subunit deficiency in two Japanese children with different phenotypes of glutaric acidemia type II.

PubMed

Purevjav, E; Kimura, M; Takusa, Y; Ohura, T; Tsuchiya, M; Hara, N; Fukao, T; Yamaguchi, S

2002-09-01

Electron transfer flavoprotein is a mitochondrial matrix protein composed of alpha- and beta-subunits (ETF alpha and ETF beta, respectively). This protein transfers electrons between several mitochondrial dehydrogenases and the main respiratory chain via ETF dehydrogenase (ETF-DH). Defects in ETF or ETF-DH cause glutaric acidemias type II (GAII). We investigated the molecular basis of ETF alpha deficiency in two Japanese children with different clinical phenotypes using expression study. Patient 1 had the severe form of GAII, a compound heterozygote of two mutations: 799G to A (alpha G267R) and nonsense 7C to T (alpha R3X). Patient 2 had the mild form and carried two heterozygous mutations: 764G to T (alpha G255V) and 478delG (frameshift). Both patients had one each of missense mutations in one allele; the others were either nonsense or truncated. Restriction enzyme digestion assay using genomic DNAs from 100 healthy Japanese revealed that these mutations were all novel. No signal for ETF alpha was detected by immunoblotting in cases of missense mutants, while wild-type cDNA resulted in expression of ETF alpha protein. Transfection with wild-type ETF alpha cDNA into cultured cells from both patients elevated incorporation of radioisotope-labelled fatty acids. These four mutations were pathogenic for GAII and missense mutations, alpha G255V and alpha G267R were considered anecdotal for mild and severe forms, respectively.

A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

McIntire, W.S.; Wemmer, D.E.; Chistoserdov, A.

Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectralmore » data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.« less

Identification of five reptile egg whites protein using MALDI-TOF mass spectrometry and LC/MS-MS analysis.

PubMed

Prajanban, Bung-on; Shawsuan, Laoo; Daduang, Sakda; Kommanee, Jintana; Roytrakul, Sittiruk; Dhiravisit, Apisak; Thammasirirak, Sompong

2012-03-16

Proteomics of egg white proteins of five reptile species, namely Siamese crocodile (Crocodylus siamensis), soft-shelled turtle (Trionyx sinensis taiwanese), red-eared slider turtle (Trachemys scripta elegans), hawksbill turtle (Eretmochelys imbricate) and green turtle (Chelonia mydas) were studied by 2D-PAGE using IPG strip pH 4-7 size 7 cm and IPG strip pH 3-10 size 24 cm. The protein spots in the egg white of the five reptile species were identified by MALDI-TOF mass spectrometry and LC/MS-MS analysis. Sequence comparison with the database revealed that reptile egg white contained at least seven protein groups, such as serpine, transferrin precursor/iron binding protein, lysozyme C, teneurin-2 (fragment), interferon-induced GTP-binding protein Mx, succinate dehydrogenase iron-sulfur subunit and olfactory receptor 46. This report confirms that transferrin precursor/iron binding protein is the major component in reptile egg white. In egg white of Siamese crocodile, twenty isoforms of transferrin precursor were found. Iron binding protein was found in four species of turtle. In egg white of soft-shelled turtle, ten isoforms of lysozyme were found. Apart from well-known reptile egg white constituents, this study identified some reptile egg white proteins, such as the teneurin-2 (fragment), the interferon-induced GTP-binding protein Mx, the olfactory receptor 46 and the succinate dehydrogenase iron-sulfur subunit. Copyright © 2012 Elsevier B.V. All rights reserved.

Modulation of NFKB1/p50 by ROS leads to impaired ATP production during MI compared to cardiac hypertrophy.

PubMed

Mitra, Arkadeep; Datta, Ritwik; Rana, Santanu; Sarkar, Sagartirtha

2018-02-01

Pathological hypertrophy and myocardial infarction (MI) are two etiologically different cardiac disorders having differential molecular mechanisms of disease manifestation. However, no study has been conducted so far to analyze and compare the differential status of energy metabolism in these two disease forms. It was shown recently by our group that production of ATP is significantly impaired during MI along with inhibition of pyruvate dehydrogenase E1-β (PDHE1 B) by pyruvate dehydrogenase kinase 4 (PDK4). However, the ATP levels showed no significant change during pathological hypertrophy compared to control group. To seek a plausible explanation of this phenomenon, the peroxisome proliferator-activated receptor alpha (PPAR) pathway was studied in all the experimental groups which revealed that PGC1α- ERRα axis remains active in MI while the same remained inactive during pathological hypertrophy possibly by NF-κB that plays a significant role in deactivating this pathway during hypertrophy. At the same time, it was observed that reactive oxygen species (ROS) negatively regulates NF-κB activity during MI by oxidation of cysteine residues of p50- the DNA binding subunit of NF-κB. Thus, this study reports for the first time, a possible mechanism for the differential status of energy metabolism during two etiologically different cardiac pathophysiological conditions involving PGC1α-ERRα axis along with p50 subunit of NF-κB. © 2017 Wiley Periodicals, Inc.

Therapeutic potential of Mediator complex subunits in metabolic diseases.

PubMed

Ranjan, Amol; Ansari, Suraiya A

2018-01-01

The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

PubMed

Puentes-Cala, Edinson; Liebeke, Manuel; Markert, Stephanie; Harder, Jens

2018-05-01

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprene and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a dithiothreitol:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism ( ctm ) in Castellaniella defragrans and were annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB and CtmAB in E. coli demonstrated that limonene dehydrogenase activity required both subunits carrying each a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest a placement in EC 1.17.99.8. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclohexanecarboxyl-Coenzyme A (CoA) and Cyclohex-1-ene-1-Carboxyl-CoA Dehydrogenases, Two Enzymes Involved in the Fermentation of Benzoate and Crotonate in Syntrophus aciditrophicus

PubMed Central

Kung, Johannes W.; Seifert, Jana; von Bergen, Martin

2013-01-01

The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation. PMID:23667239

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

PubMed

Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

2010-03-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Genetic homogeneity of Fascioloides magna in Austria.

PubMed

Husch, Christian; Sattmann, Helmut; Hörweg, Christoph; Ursprung, Josef; Walochnik, Julia

2017-08-30

The large American liver fluke, Fascioloides magna, is an economically relevant parasite of both domestic and wild ungulates. F. magna was repeatedly introduced into Europe, for the first time already in the 19th century. In Austria, a stable population of F. magna has established in the Danube floodplain forests southeast of Vienna. The aim of this study was to determine the genetic diversity of F. magna in Austria. A total of 26 individuals from various regions within the known area of distribution were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene haplotypes. Interestingly, all 26 individuals revealed one and the same haplotype, namely concatenated haplotype Ha5. This indicates a homogenous population of F. magna in Austria and may argue for a single introduction. Alternatively, genetic homogeneity might also be explained by a bottleneck effect and/or genetic drift. Copyright © 2017 Elsevier B.V. All rights reserved.

The alpaca (Vicugna pacos) as a natural intermediate host of Taenia omissa (Cestoda: Taeniidae).

PubMed

Gomez-Puerta, Luis A; Yucra, Dora; Lopez-Urbina, Maria T; Gonzalez, Armando E

2017-11-15

Three metacestodes were collected from the mesentery and the surface of the liver of three adult alpacas (Vicugna pacos) in a slaughterhouse located in Puno, Peru. Various features of the metacestodes were observed for morphological identification. A molecular diagnosis was performed by PCR-based sequencing of mitochondrial genes of cytochrome c oxidase subunit 1 (cox1) and the NADH dehydrogenase subunit 1 (nad1). All metacestodes were identified as Taenia omissa by morphology and molecular methods The isolates from alpacas showed significant sequence similarity with previously reported isolates of T. omissa (95.7-98.1% in cox1 and 94.6-95.1% in nad1). Our report is the first to detect T. omissa metacestodes in alpacas and to reveal that alpacas are natural intermediate hosts for this parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

Designing non-native iron-binding site on a protein cage for biological synthesis of nanoparticles.

PubMed

Peng, Tao; Paramelle, David; Sana, Barindra; Lee, Chiu Fan; Lim, Sierin

2014-08-13

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

PubMed

Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

2014-10-20

We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

PubMed Central

Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host.

PubMed

Iwata, Fumiko; Shinjyo, Noriko; Amino, Hisako; Sakamoto, Kimitoshi; Islam, M Khyrul; Tsuji, Naotoshi; Kita, Kiyoshi

2008-03-01

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.

Amino acid sequence of the human fibronectin receptor

PubMed Central

1987-01-01

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+- binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors. PMID:2958481

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

PubMed

Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

2015-01-13

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

Allosteric mechanism controls traffic in the chaperone/usher pathway.

PubMed

Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V

2012-11-07

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Building a pseudo-atomic model of the anaphase-promoting complex.

PubMed

Kulkarni, Kiran; Zhang, Ziguo; Chang, Leifu; Yang, Jing; da Fonseca, Paula C A; Barford, David

2013-11-01

The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14-15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

PubMed

Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Lucchi, Chiara; Correia-de-Sá, Paulo; Videira, Arnaldo

2015-10-01

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics. Copyright © 2015 Elsevier B.V. All rights reserved.

The Thiamine-Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2004-01-01

Thiamin pyrophosphate (TPP), a derivative of vitamin B1, is a cofactor for enzymes performing catalysis in pathways of energy production including the well known decarboxylation of a-keto acid dehydrogenases followed by transketolation. TPP-dependent enzymes constitute a structurally and functionally diverse group exhibiting multimeric subunit organization, multiple domains and two chemically equivalent catalytic centers. Annotation of functional TPP-dependcnt enzymes, therefore, has not been trivial due to low sequence similarity related to this complex organization. Our approach to analysis of structures of known TPP-dependent enzymes reveals for the first time features common to this group, which we have termed the TPP-motif. The TPP-motif consists of specific spatial arrangements of structural elements and their specific contacts to provide for a flip-flop, or alternate site, enzymatic mechanism of action. Analysis of structural elements entrained in the flip-flop action displayed by TPP-dependent enzymes reveals a novel definition of the common amino acid sequences. These sequences allow for annotation of TPP-dependent enzymes, thus advancing functional proteomics. Further details of three-dimensional structures of TPP-dependent enzymes will be discussed.

Dopamine-Secreting Paraganglioma in the Retroperitoneum.

PubMed

Matsuda, Yusuke; Kimura, Noriko; Yoshimoto, Takanobu; Sekiguchi, Yoshihiro; Tomoishi, Junzo; Kasahara, Ichiro; Hara, Yoshihito; Ogawa, Yoshihiro

2017-03-01

Pheochromocytomas and paragangliomas, which exclusively produce dopamine, are very rare. Herein, we report for the first time a Japanese case of an exclusively dopamine-producing paraganglioma accompanied by detailed immunohistochemical analyses. A 70-year-old Japanese woman was referred to our hospital for functional examination of her left retroperitoneal mass. Her adrenal functions were normal, except for excessive dopamine secretion. After the tumorectomy, her dopamine level normalized. The histopathological diagnosis of the tumor was paraganglioma; this was confirmed by positive immunostaining of chromogranin A (CgA), tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and succinate dehydrogenase gene subunit B (SDHB). However, the immunostaining of CgA in the tumor cells showed peculiar dot-like staining located corresponding to Golgi complex in the perinuclear area, rather than the diffuse cytoplasmic staining usually observed in epinephrine- or norepinephrine-producing functional pheochromocytomas and paragangliomas. The immunohistochemical results suggested that the tumor cells had sparse neuroendocrine granules in the cytoplasm, resulting in inhibition of catecholamine synthesis from dopamine to norepinephrine in neurosecretory granules. This may be the mechanism responsible for exclusive dopamine secretion in the present case.

Mode of action of the qcr9 and cat3 mutations in restoring the ability of Saccharomyces cerevisiae tps1 mutants to grow on glucose.

PubMed

Blázquez, M A; Gancedo, C

1995-12-20

Mutations in the TPS1 gene, which encodes trehalose-6-P synthase, cause a glucose-negative phenotype in Saccharomyces cerevisiae. Antimycin A or disruption of the QCR9 gene, which encodes one subunit of the cytochrome bc1 complex, restore the ability to grow in glucose-containing media. Under these conditions the cell excreted a large amount of glycerol, corresponding to about 20% of the glucose taken up. Suppression appears to be achieved by diversion of accumulated glycolytic intermediates to the production of glycerol, thereby providing NAD+ and phosphate for the glyceraldehyde-3-P dehydrogenase reaction. Analysis of the mutation sci1-1, which also suppresses the glucose-negative phenotype of tps1 mutants, showed that glucose transport was decreased in sci1-1 mutants. The gene SCI1 was cloned and its nucleotide sequence revealed it to be identical to CAT3/SNF4. The suppression mediated by sci1-1 is attributable to a decrease in glycolytic flux.

Limited mitogenomic degradation in response to a parasitic lifestyle in Orobanchaceae

PubMed Central

Fan, Weishu; Zhu, Andan; Kozaczek, Melisa; Shah, Neethu; Pabón-Mora, Natalia; González, Favio; Mower, Jeffrey P.

2016-01-01

In parasitic plants, the reduction in plastid genome (plastome) size and content is driven predominantly by the loss of photosynthetic genes. The first completed mitochondrial genomes (mitogenomes) from parasitic mistletoes also exhibit significant degradation, but the generality of this observation for other parasitic plants is unclear. We sequenced the complete mitogenome and plastome of the hemiparasite Castilleja paramensis (Orobanchaceae) and compared them with additional holoparasitic, hemiparasitic and nonparasitic species from Orobanchaceae. Comparative mitogenomic analysis revealed minimal gene loss among the seven Orobanchaceae species, indicating the retention of typical mitochondrial function among Orobanchaceae species. Phylogenetic analysis demonstrated that the mobile cox1 intron was acquired vertically from a nonparasitic ancestor, arguing against a role for Orobanchaceae parasites in the horizontal acquisition or distribution of this intron. The C. paramensis plastome has retained nearly all genes except for the recent pseudogenization of four subunits of the NAD(P)H dehydrogenase complex, indicating a very early stage of plastome degradation. These results lend support to the notion that loss of ndh gene function is the first step of plastome degradation in the transition to a parasitic lifestyle. PMID:27808159

Voltage-gated sodium channel β subunits: The power outside the pore in brain development and disease.

PubMed

Hull, Jacob M; Isom, Lori L

2018-04-01

Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and β subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two β subunits; a noncovalently linked β1 or β3 and a covalently linked β2 or β4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC β subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of β1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC β subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction. This article is part of the Special Issue entitled 'Channelopathies.' Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides*

PubMed Central

Nadeau, Owen W.; Anderson, David W.; Yang, Qing; Artigues, Antonio; Paschall, Justin E.; Wyckoff, Gerald J.; McClintock, Jennifer L.; Carlson, Gerald M.

2007-01-01

Phosphorylase kinase (PhK), an (αβγδ)4 complex, regulates glycogenolysis. Its activity, catalyzed by the γ subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory α, β and δ subunits. Activation of the catalytic γ subunit in the PhK complex by phosphorylation is known to be predominantly mediated by the regulatory β subunit, which undergoes a conformational change that is structurally linked with the γ subunit and that is characterized by the ability to form β-β dimers using a short chemical crosslinker. To determine potential regions of interaction of the β and γ subunits, we have used chemical crosslinking and 2-hybrid screening. The β and γ subunits were chemically crosslinked to each other in phosphorylated PhK, and crosslinked peptides were identified in digests of the kinase by Fourier transform mass spectrometry in combination with a search engine developed ‘in house’ that generates a hypothetical list of crosslinked peptides. Such a conjugate between β and γ was identified, verified by MS/MS and shown to correspond to crosslinking between K303 in the C-terminal regulatory domain of γ (γCRD) and R18 in the N-terminal regulatory region of β (β1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of β inhibited the crosslinking between β and γ in the complex, and was itself crosslinked to K303 of γ. Through the use of 2-hybrid screening, the β1-31 region was also shown to control β subunit self-interactions, which were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the β and γ subunits. The sum of our results considered together with previous findings implicates the γCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme’s regulatory subunits and is proximal to the active site cleft. PMID:17123541

G protein βγ11 complex translocation is induced by Gi, Gq and Gs coupling receptors and is regulated by the α subunit type

PubMed Central

Azpiazu, Inaki; Akgoz, Muslum; Kalyanaraman, Vani; Gautam, N.

2008-01-01

G protein activation by Gi/Go coupling M2 muscarinic receptors, Gq coupling M3 receptors and Gs coupling β2 adrenergic receptors causes rapid reversible translocation of the G protein γ11 subunit from the plasma membrane to the Golgi complex. Co-translocation of the β1 subunit suggests that γ11 translocates as a βγ complex. Pertussis toxin ADP ribosylation of the αi subunit type or substitution of the C terminal domain of αo with the corresponding region of αs inhibits γ11 translocation demonstrating that α subunit interaction with a receptor and its activation are requirements for the translocation. The rate of γ11 translocation is sensitive to the rate of activation of the G protein α subunit. α subunit types that show high receptor activated rates of guanine nucleotide exchange in vitro support high rates of γ11 translocation compared to α subunit types that have a relatively lower rate of guanine nucleotide exchange. The results suggest that the receptor induced translocation of γ11 is controlled by the rate of cycling of the G protein through active and inactive forms. They also demonstrate that imaging of γ11 translocation can be used as a non-invasive tool to measure the relative activities of wild type or mutant receptor and α subunit types in a live cell. PMID:16242307

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

NASA Technical Reports Server (NTRS)

Henry, R. L.; Takemoto, L. J.; Murphy, J.; Gallegos, G. L.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine.

PubMed

Volkan, Ender; Ford, Bradley A; Pinkner, Jerome S; Dodson, Karen W; Henderson, Nadine S; Thanassi, David G; Waksman, Gabriel; Hultgren, Scott J

2012-06-12

P pili are prototypical chaperone-usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesin-bound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTD-Plug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus.

Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

PubMed Central

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.

2014-01-01

ABSTRACT The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex. PMID:25352624

The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

PubMed

Kück, Ulrich; Beier, Anna M; Teichert, Ines

2016-05-01

The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

l-Valine Production during Growth of Pyruvate Dehydrogenase Complex- Deficient Corynebacterium glutamicum in the Presence of Ethanol or by Inactivation of the Transcriptional Regulator SugR▿

PubMed Central

Blombach, Bastian; Arndt, Annette; Auchter, Marc; Eikmanns, Bernhard J.

2009-01-01

Pyruvate dehydrogenase complex-deficient strains of Corynebacterium glutamicum produce l-valine from glucose only after depletion of the acetate required for growth. Here we show that inactivation of the DeoR-type transcriptional regulator SugR or replacement of acetate by ethanol already in course of the growth phase results in efficient l-valine production. PMID:19088318

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation*

PubMed Central

Letts, James A.; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A.

2016-01-01

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. PMID:27672209

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1.

PubMed

Iizuka, Ryo; Sugano, Yuri; Ide, Naoki; Ohtaki, Akashi; Yoshida, Takao; Fujiwara, Shinsuke; Imanaka, Tadayuki; Yohda, Masafumi

2008-03-28

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two alpha subunits and four beta subunits with the structure of a double beta-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (alpha1, alpha2, beta1, and beta2) from T. KS-1. All of them (alpha1-beta1, alpha2-beta1, alpha1-beta2, and alpha2-beta2) exist as alpha(2)beta(4) heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the beta1 subunit interacted with the chaperonins more strongly than those with the beta2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.

Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis.

PubMed

Wang, Huping; Han, Wenyu; Takagi, Junichi; Cong, Yao

2018-05-11

Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biochemical basis of type IB (E1beta ) mutations in maple syrup urine disease. A prevalent allele in patients from the Druze kindred in Israel.

PubMed

Wynn, R M; Chuang, J L; Sansaricq, C; Mandel, H; Chuang, D T

2001-09-28

Maple syrup urine disease (MSUD) is a metabolic disorder associated with often-fatal ketoacidosis, neurological derangement, and mental retardation. In this study, we identify and characterize two novel type IB MSUD mutations in Israeli patients, which affect the E1beta subunit in the decarboxylase (E1) component of the branched-chain alpha-ketoacid dehydrogenase complex. The recombinant mutant E1 carrying the prevalent S289L-beta (TCG --> TTG) mutation in the Druze kindred exists as a stable inactive alphabeta heterodimer. Based on the human E1 structure, the S289L-beta mutation disrupts the interactions between Ser-289-beta and Glu-290-beta', and between Arg-309-beta and Glu-290-beta', which are essential for native alpha(2)beta(2) heterotetrameric assembly. The R133P-beta (CGG --> CCG) mutation, on the other hand, is inefficiently expressed in Escherichia coli as heterotetramers in a temperature-dependent manner. The R133P-beta mutant E1 exhibits significant residual activity but is markedly less stable than the wild-type, as measured by thermal inactivation and free energy change of denaturation. The R133P-beta substitution abrogates the coordination of Arg-133-beta to Ala-95-beta, Glu-96-beta, and Ile-97-beta, which is important for strand-strand interactions and K(+) ion binding in the beta subunit. These findings provide new insights into folding and assembly of human E1 and will facilitate DNA-based diagnosis for MSUD in the Israeli population.

Phylogenetic Status of an Unrecorded Species of Curvularia, C. spicifera, Based on Current Classification System of Curvularia and Bipolaris Group Using Multi Loci.

PubMed

Jeon, Sun Jeong; Nguyen, Thi Thuong Thuong; Lee, Hyang Burm

2015-09-01

A seed-borne fungus, Curvularia sp. EML-KWD01, was isolated from an indigenous wheat seed by standard blotter method. This fungus was characterized based on the morphological characteristics and molecular phylogenetic analysis. Phylogenetic status of the fungus was determined using sequences of three loci: rDNA internal transcribed spacer, large ribosomal subunit, and glyceraldehyde 3-phosphate dehydrogenase gene. Multi loci sequencing analysis revealed that this fungus was Curvularia spicifera within Curvularia group 2 of family Pleosporaceae.

Genetics of bacteria that utilize one carbon compounds: Final report, March 1, 1982-February 29, 1988

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanson, R.S.

Broad host range plasmid vectors useful for cloning genes from bacteria that grow on methane and methanol were constructed. We have cloned and mapped nineteen genes required for the growth of Methylobacterium organophilum strain XX on methanol. Nineteen genes were found in seven linkage groups on the M. organophilum genome and were separated by 40 kb or more. Eleven genes were required for the synthesis of methanol dehydrogenase (MDH) and were located in three unlinked gene clusters. The MDH structural gene was localized on a 2.5 kb DNA fragment. The gene was sequenced and contains a 175 bp untranslated leadermore » sequence, a signal sequence and the structural gene. MDH messenger RNA (mRNA) has a half life of approximately 20 min. and is present at approximately 2% of the cellular mRNA. The structural gene for the ..gamma.. subunit of methane monoxygenases has been cloned from Methylosporovibrio. Methane monooxygenase subunits have been purified by Prof. J. Lipscomb's laboratory and are being sequenced to construct DNA probes to identify cloned subunit genes. New facultative methylotrophic bacteria were isolated and characterized. Several amino acid auxotrophs have been isolated. 11 refs.« less

The plant Polycomb repressive complex 1 (PRC1) existed in the ancestor of seed plants and has a complex duplication history.

PubMed

Berke, Lidija; Snel, Berend

2015-03-13

Polycomb repressive complex 1 (PRC1) is an essential protein complex for plant development. It catalyzes ubiquitination of histone H2A that is an important part of the transcription repression machinery. Absence of PRC1 subunits in Arabidopsis thaliana plants causes severe developmental defects. Many aspects of the plant PRC1 are elusive, including its origin and phylogenetic distribution. We established the evolutionary history of the plant PRC1 subunits (LHP1, Ring1a-b, Bmi1a-c, EMF1, and VRN1), enabled by sensitive phylogenetic methods and newly sequenced plant genomes from previously unsampled taxonomic groups. We showed that all PRC1 core subunits exist in gymnosperms, earlier than previously thought, and that VRN1 is a recent addition, found exclusively in eudicots. The retention of individual subunits in chlorophytes, mosses, lycophytes and monilophytes indicates that they can moonlight as part of other complexes or processes. Moreover, we showed that most PRC1 subunits underwent a complex, duplication-rich history that differs significantly between Brassicaceae and other eudicots. PRC1 existed in the last common ancestor of seed plants where it likely played an important regulatory role, aiding their radiation. The presence of LHP1, Ring1 and Bmi1 in mosses, lycophytes and monilophytes also suggests the presence of a primitive yet functional PRC1.

The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

PubMed

Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

2014-01-01

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

Influence of Coulombic repulsion on the dissociation pathways and energetics of multiprotein complexes in the gas phase.

PubMed

Sinelnikov, Igor; Kitova, Elena N; Klassen, John S

2007-04-01

Thermal dissociation experiments, implemented with blackbody infrared radiative dissociation and Fourier-transform ion cyclotron resonance mass spectrometry, are performed on gaseous protonated and deprotonated ions of the homopentameric B subunits of Shiga toxin 1 (Stx1 B5) and Shiga toxin 2 (Stx2 B5) and the homotetramer streptavidin (S4). Dissociation of the gaseous, multisubunit complexes proceeds predominantly by the loss of a single subunit. Notably, the fractional partitioning of charge between the product ions, i.e., the leaving subunit and the resulting multimer, for a given complex is, within error, constant over the range of charge states investigated. The Arrhenius activation parameters (E(a), A) measured for the loss of subunit decrease with increasing charge state of the complex. However, the parameters for the protonated and deprotonated ions, with the same number of charges, are indistinguishable. The influence of the complex charge state on the dissociation pathways and the magnitude of the dissociation E(a) are modeled theoretically with the discrete charge droplet model (DCDM) and the protein structure model (PSM), wherein the structure of the subunits is considered. Importantly, the major subunit charge states observed experimentally for the Stx1 B5(n+/-) ions correspond to the minimum energy charge distribution predicted by DCDM and PSM assuming a late dissociative transition-state (TS); while for structurally-related Stx2 B5(n+) ions, the experimental charge distribution corresponds to an early TS. It is proposed that the lateness of the TS is related, in part, to the degree of unfolding of the leaving subunit, with Stx1 B being more unfolded than Stx2 B. PSM, incorporating significant subunit unfolding is necessary to account for the product ions observed for the S4(n+) ions. The contribution of Coulombic repulsion to the dissociation E(a) is quantified and the intrinsic activation energy is estimated for the first time.

Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein

PubMed Central

2012-01-01

Background Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150Glued), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11). Results To reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons. Conclusions The dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events. PMID:22726940

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

PubMed Central

Anandhakumar, Jayamani; Moustafa, Yara W.; Chowdhary, Surabhi; Kainth, Amoldeep S.

2016-01-01

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the “anchor away” (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. PMID:27185874

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

PubMed

Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

2017-04-01

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome.

PubMed Central

Serino, G; Tsuge, T; Kwok, S; Matsui, M; Wei, N; Deng, X W

1999-01-01

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome. PMID:10521526

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

PubMed Central

Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

2015-01-01

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-04-01

The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

Evidence that assembly of the yeast cytochrome bc1 complex involves formation of a large core structure in the inner mitochondrial membrane

PubMed Central

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.

2009-01-01

The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

A Family of G Protein βγ Subunits Translocate Reversibly from the Plasma Membrane to Endomembranes on Receptor Activation*S

PubMed Central

Saini, Deepak Kumar; Kalyanaraman, Vani; Chisari, Mariangela; Gautam, Narasimhan

2008-01-01

The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein γ subunit family translocate specifically from the PM to endomembranes. The γ subunits translocate as βγ complexes, whereas the α subunit is retained on the PM. Depending on the γ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the γ subunit type. Different γ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various γ subunits and their translocation properties. γ subunits with similar translocation properties are more closely related to each other. Consistent with this relationship, introducing residues conserved in translocating subunits into a non-translocating subunit results in a gain of function. Inhibitors of vesicle-mediated trafficking and palmitoylation suggest that translocation is diffusion-mediated and controlled by acylation similar to the shuttling of G protein subunits (Chisari, M., Saini, D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem. 282, 24092–24098). These results suggest that the continual testing of cytosolic surfaces of cell membranes by G protein subunits facilitates an activated cell surface receptor to direct potentially active G protein βγ subunits to intracellular membranes. PMID:17581822

Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-07-01

The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

2003-01-01

The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.

Phellinus rimosus improves mitochondrial energy status and attenuates nephrotoxicity in diabetic rats.

PubMed

Rony, K A; Ajith, T A; Kuttikadan, Tony A; Blaze, R; Janardhanan, K K

2017-09-26

Mitochondrial dysfunction and increase in reactive oxygen species during diabetes can lead to pathological consequences in kidneys. The present study was aimed to investigate the effect of Phellinus rimosus in the streptozotocin (STZ)-induced diabetic rat renal mitochondria and the possible mechanism of protection. Phellinus rimosus (50 and 250 mg/kg, p.o) was treated after inducing diabetes by STZ (45 mg/kg, i.p) in rats. The serum samples were subjected to creatinine and urea estimation. Mitochondrial antioxidant status such as mitochondrial superoxide dismutase, glutathione peroxidase, and reduced glutathione; adenosine triphosphate level; and lipid peroxidation were measured. The activities of Krebs cycle enzymes such as isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, III, and IV in kidney mitochondria were also determined. Administration of P. rimosus (250 mg/kg b.wt) once daily for 30 days, significantly (p<0.05) enhanced the activities of Krebs cycle dehydrogenases, mitochondrial electron transport chain complexes, and ATP level. Further, P. rimosus had significantly protected the renal mitochondrial antioxidant status and lipid peroxidation. The results of the study concluded that by limiting the extent of renal mitochondrial damage in the hyperglycemic state, P. rimosus alleviated nephrotoxicity.

Exercise training improves vascular mitochondrial function

PubMed Central

Park, Song-Young; Rossman, Matthew J.; Gifford, Jayson R.; Bharath, Leena P.; Bauersachs, Johann; Richardson, Russell S.; Abel, E. Dale; Symons, J. David

2016-01-01

Exercise training is recognized to improve cardiac and skeletal muscle mitochondrial respiratory capacity; however, the impact of chronic exercise on vascular mitochondrial respiratory function is unknown. We hypothesized that exercise training concomitantly increases both vascular mitochondrial respiratory capacity and vascular function. Arteries from both sedentary (SED) and swim-trained (EX, 5 wk) mice were compared in terms of mitochondrial respiratory function, mitochondrial content, markers of mitochondrial biogenesis, redox balance, nitric oxide (NO) signaling, and vessel function. Mitochondrial complex I and complex I + II state 3 respiration and the respiratory control ratio (complex I + II state 3 respiration/complex I state 2 respiration) were greater in vessels from EX relative to SED mice, despite similar levels of arterial citrate synthase activity and mitochondrial DNA content. Furthermore, compared with the SED mice, arteries from EX mice displayed elevated transcript levels of peroxisome proliferative activated receptor-γ coactivator-1α and the downstream targets cytochrome c oxidase subunit IV isoform 1, isocitrate dehydrogenase (Idh) 2, and Idh3a, increased manganese superoxide dismutase protein expression, increased endothelial NO synthase phosphorylation (Ser1177), and suppressed reactive oxygen species generation (all P < 0.05). Although there were no differences in EX and SED mice concerning endothelium-dependent and endothelium-independent vasorelaxation, phenylephrine-induced vasocontraction was blunted in vessels from EX compared with SED mice, and this effect was normalized by NOS inhibition. These training-induced increases in vascular mitochondrial respiratory capacity and evidence of improved redox balance, which may, at least in part, be attributable to elevated NO bioavailability, have the potential to protect against age- and disease-related challenges to arterial function. PMID:26825520

N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex.

PubMed

Wu, Wei-Hua; Wu, Chwen-Huey; Ladurner, Andreas; Mizuguchi, Gaku; Wei, Debbie; Xiao, Hua; Luk, Ed; Ranjan, Anand; Wu, Carl

2009-03-06

Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

Short-term regulation of the alpha-ketoglutarate dehydrogenase complex by energy-linked and some other effectors.

PubMed

Strumilo, S

2005-07-01

The question of regulation of alpha-ketoglutarate dehydrogenase complex (KGDHC) has been considered in the biochemical literature very rarely. Moreover, such information is not usually accurate, especially in biochemical textbooks. From the mini-review of research works published during the last 25 years, the following basic view is clear: a) animal KGDHC is very sensitive to ADP, P(i), and Ca2+; b) these positive effectors increase manifold the affinity of KGDHC to alpha-ketoglutarate; c) KGDHC is inhibited by ATP, NADH, and succinyl-CoA; d) the ATP effect is realized in several ways, probably mainly via opposition versus ADP activation; e) NADH, besides inhibiting dihydrolipoamide dehydrogenase component competitively versus NAD+, decreases the affinity of alpha-ketoglutarate dehydrogenase to substrate and inactivates it; f) thioredoxin protects KGDHC from self-inactivation during catalysis; g) bacterial and plant KGDHC is activated by AMP instead of ADP. These main effects form the basis of short-term regulation of KGDHC.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in the subunit stoichiometry study of high-mass non-covalent complexes

NASA Astrophysics Data System (ADS)

Moniatte, M.; Lesieur, C.; Vecsey-Semjen, B.; Buckley, J. T.; Pattus, F.; van der Goot, F. G.; van Dorsselaer, A.

1997-12-01

This study explores the potential of MALDI-TOF MS for the mass measurement of large non-covalent protein complexes. The following non-covalent complexes have been investigated: aerolysin from Aeromonas hydrophila (335 kDa) and [alpha]-haemolysin from Staphylococcus aureus (233 kDa) which are both cytolytic toxins, three enzymes known to be homotetramers in solution: bovine liver catalase (235 kDa), rabbit muscle pyruvate kinase (232 kDa), yeast alcohol dehydrogenase (147 kDa) and finally a lectin, concanavalin A (102 kDa). Three different matrix preparations were systematically tested under various conditions: ferulic acid dissolved in THF, 2,6-dihydroxyacetophenone in 20 mM aqueous ammonium citrate and a two-step sample preparation with sinapinic acid. It was possible to find a suitable combination of matrix and preparation type which allowed the molecularity of all complexes tested to be deduced from the MALDI mass spectrum. Trimeric and tetrameric intermediates accumulating during the formation of the active heptameric aerolysin complex were also identified, this allowing a formation mechanism to be proposed. The observation of large specific non-covalent complexes has been found to be dependent on the choice of matrix, the type of sample preparation used, the solvent evaporation speed, the pH of the resulting matrix-sample mixture and the number of shots acquired on a given area. From this set of experiments, some useful guidelines for the observation of large complexes by MALDI could therefore be deduced. Fast evaporation of the solvent is particularly necessary in the case of pH sensitive complexes. An ESMS study on the same non-covalent complexes indicated that, rather surprisingly, reliable results could be obtained by MALDI-TOF MS on several very large complexes (above 200 kDa) for which ESMS yielded no clear spectra.

LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes.

PubMed

Mund, Markus; Overbeck, Jan H; Ullmann, Janina; Sprangers, Remco

2013-10-18

Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of Creative Commons the Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

PubMed

Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

2016-06-21

The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood. The first fungal STRIPAK was described in Sordaria macrospora, which is a well-established model organism used to study the formation of fungal fruiting bodies, three-dimensional organ-like structures. We analyzed STRIPAK subunit PP2Ac1, catalytic subunit 1 of protein phosphatase PP2A, to study the importance of the catalytic activity of this protein during sexual development. The results of our yeast two-hybrid analysis and tandem affinity purification, followed by mass spectrometry, indicate that PP2Ac1 activity connects STRIPAK with other signaling pathways and thus forms a large interconnected signaling network. Copyright © 2016 Beier et al.

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

PubMed Central

Akgoz, Muslum; Kalyanaraman, Vani; Gautam, N.

2008-01-01

On activation of a receptor the G protein βγ complex translocates away from the receptor on the plasma membrane to the Golgi complex. The rate of translocation is influenced by the type of γ subunit associated with the G protein. Complementary approaches — imaging living cells expressing fluorescent protein tagged G proteins and assaying reconstituted receptors and G proteins in vitro — were used to identify mechanisms at the basis of the translocation process. Translocation of Gβγ containing mutant γ subunits with altered prenyl moieties showed that the differences in the prenyl moieties were not sufficient to explain the differential effects of geranylgeranylated γ5 and farnesylated γ11 on the translocation process. The translocation properties of Gβγ were altered dramatically by mutating the C terminal tail region of the γ subunit. The translocation characteristics of these mutants suggest that after receptor activation, Gβγ retains contact with a receptor through the γ subunit C terminal domain and that differential interaction of the activated receptor with this domain controls Gβγ translocation from the plasma membrane. PMID:16517125

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

2010-01-01

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

PubMed

Palù, Giorgio; Loregian, Arianna

2013-09-01

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET.

PubMed

Carrasquel-Ursulaez, Willy; Alvarez, Osvaldo; Bezanilla, Francisco; Latorre, Ramon

2018-06-05

Two families of accessory proteins, β and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca 2+ , the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb +3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

PubMed

Halimi, Yair; Dessau, Moshe; Pollak, Shaul; Ast, Tslil; Erez, Tamir; Livnat-Levanon, Nurit; Karniol, Baruch; Hirsch, Joel A; Chamovitz, Daniel A

2011-09-01

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

Genetic analysis of the cytoplasmic dynein subunit families.

PubMed

Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic Analysis of the Cytoplasmic Dynein Subunit Families

PubMed Central

Pfister, K. Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M. C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

PubMed

Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

2017-05-01

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP + reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

PubMed

Droux, M; Miginiac-Maslow, M; Jacquot, J P; Gadal, P; Crawford, N A; Kosower, N S; Buchanan, B B

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Properties of lactate dehydrogenase from the isopod, Saduria entomon.

PubMed

Mulkiewicz, E; Zietara, M S; Stachowiak, K; Skorkowski, E F

2000-07-01

Saduria entomon lactate dehydrogenase (LDH-A4*) from thorax muscle was purified about 89 fold to specific activity 510 micromol NADH/min/mg using Cibacron Blue 3GA Agarose and Oxamate-Agarose chromatographies. The enzyme is a tetramer, with molecular weight of 140 kDa for the native enzyme and 36 kDa for the subunit. The isoelectric point was at pH 5.7. The enzyme possesses high heat stability (T50 = 71.5 degrees C). The optimum pH for pyruvate reduction reaction was 6.5, while for lactate oxidation one, the maximum activity was at pH 9.1. The Km for pyruvate was minimal at 5 degrees C, the average environmental temperature of the isopod. The Km values determined at 30 degrees C and optimal pH for pyruvate reduction and lactate oxidation were 0.18 and 90.04 mM, respectively. Amino acid compositional analyses showed the strongest resemblance of the isopod isoenzyme to cod (Gadus morhua) LDH-C4.

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue.

PubMed

Rovira, Alexander R; Fin, Andrea; Tor, Yitzhak

2017-11-08

The synthesis, photophysics, and biochemical utility of a fluorescent NAD + analogue based on an isothiazolo[4,3-d]pyrimidine core (N tz AD + ) are described. Enzymatic reactions, photophysically monitored in real time, show N tz AD + and N tz ADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N tz AD + is converted to N tz ADH, reflecting a complementary photophysical behavior to that of the native NAD + /NADH. N tz AD + and N tz ADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tz ADP-ribose. N tz AD + also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tz ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD + .

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn,more » reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.« less

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from the leaf tissues of a basin mangrove Lumnitzera racemosa Willd.

PubMed

Murugan, K; Arunkumar, N S; Mohankumar, C

2004-01-01

Cinnamyl alcohol-NADPH-dehydrogenase (CAD), the marker enzyme of lignin biosynthesis was purified from the leaf tissues of a basin mangrove Lumnitzera racemosa by ammonium sulphate precipitation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the CAD enzyme was determined as 89 kDa, by size elution chromatography. SDS-PAGE of CAD revealed two closely associated bands of 45 kDa and 42 kDa as heterogenous subunits. The optimum pH of CAD was found to be 4.0. Km for the substrates cinnamaldehyde, coniferaldehyde and sinapaldehyde was determined. Cinnamaldehyde showed higher Km value than sinapaldehyde and coniferaldehyde. The correlation of activity of CAD with the amount of lignin was found less significant in L. racemosa, compared to plant species of other habitats viz., mesophytes, xerophytes and hydrophytes, suggesting that CAD possibly exhibits physiological suppression due to the saline habitat of the plant.

A METABOLIC SIGNATURE FOR LONG-LIFE IN THE C. ELEGANS MIT MUTANTS

PubMed Central

Butler, Jeffrey A.; Mishur, Robert J.; Bhaskaran, Shylesh; Rea, Shane L.

2012-01-01

SUMMARY Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent upon concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer’s disease. When the expression of DLD in wild type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened but, at higher doses, it was significantly lengthened, suggesting DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. PMID:23173729

Antisense and sense poly(A)-RNAs from the Xenopus laevis pyruvate dehydrogenase gene loci are regulated with message production during embryogenesis.

PubMed

Islam, N; Poitras, L; Gagnon, F; Moss, T

1996-10-17

The structure and temporal expression of two Xenopus cDNAs encoding the beta subunit of pyruvate dehydrogenase (XPdhE1 beta) have been determined. XPdhE1 beta was 88% homologous to mature human PdhE1 beta, but the putative N-terminal mitochondrial signal peptide was poorly conserved. Zygotic expression of XPdhE1 beta mRNA was detected at neural tube closure and increased until stage 40. RT-PCR cloning identified a short homology to a protein kinase open reading frame within the 3' non-coding sequence of the XPdhE1 beta cDNAs. This homology, which occurred on the antisense cDNA strand, was shown by strand specific RT-PCR to be transcribed in vivo as part of an antisense RNA. Northern analysis showed that this RNA formed part of an abundant and heterogeneous population of antisense and sense poly(A)-RNAs transcribed from the XPdhE1 beta loci and coordinately regulated with message production.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jiang; Malmirchegini, G. Reza; Clubb, Robert T.

Native mass spectrometry (MS) has become an invaluable tool for the characterization of proteins and non-covalent protein complexes under near physiological solution conditions. Here we report the structural characterization of human hemoglobin (Hb), a 64 kDa oxygen-transporting protein complex, by high resolution native top-down mass spectrometry using electrospray ionization (ESI) and a 15-Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Native MS preserves the non-covalent interactions between the globin subunits, and electron capture dissociation (ECD) produces fragments directly from the intact Hb complex without dissociating the subunits. Using activated ion ECD, we observe the gradual unfolding process of themore » Hb complex in the gas phase. Without protein ion activation, the native Hb shows very limited ECD fragmentation from the N-termini, suggesting a tightly packed structure of the native complex and therefore low fragmentation efficiency. Precursor ion activation allows steady increase of N-terminal fragment ions, while the C-terminal fragments remain limited (38 c ions and 4 z ions on the α chain; 36 c ions and 2 z ions on the β chain). This ECD fragmentation pattern suggests that upon activation, the Hb complex starts to unfold from the N-termini of both subunits, whereas the C-terminal regions and therefore the potential regions involved in the subunit binding interactions remain intact. ECD-MS of the Hb dimer show similar fragmentation patterns as the Hb tetramer, providing further evidence for the hypothesized unfolding process of the Hb complex in the gas phase. Native top-down ECD-MS allows efficient probing of the Hb complex structure and the subunit binding interactions in the gas phase. Finally, it may provide a fast and effective means to probe the structure of novel protein complexes that are intractable to traditional structural characterization tools.« less

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei.

PubMed

Nguyen, Tu N; Schimanski, Bernd; Zahn, André; Klumpp, Birgit; Günzl, Arthur

2006-09-01

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.

NDUFA4L2 protects against ischaemia/reperfusion-induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I.

PubMed

Li, Jianhua; Bai, Caiyan; Guo, Junxia; Liang, Wanqian; Long, Jingning

2017-07-01

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2) on myocardial ischaemia-reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia-reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT-PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl-2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down-regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R-induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl-2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down-regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury. © 2017 John Wiley & Sons Australia, Ltd.

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation.

PubMed

Letts, James A; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A

2016-11-18

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

PubMed Central

2014-01-01

Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

PubMed

Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

2014-01-01

The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

PubMed

Gordon, G L; Doelle, H W

1976-08-16

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

Fermentative Production of the Diamine Putrescine: System Metabolic Engineering of Corynebacterium Glutamicum

PubMed Central

Nguyen, Anh Q. D.; Schneider, Jens; Reddy, Gajendar Komati; Wendisch, Volker F.

2015-01-01

Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g−1·h−1 and a yield on glucose of 0.26 g·g−1. PMID:25919117

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus.

PubMed

Hektor, Harm J; Kloosterman, Harm; Dijkhuizen, Lubbert

2002-12-06

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

Short-Chain 3-Hydroxyacyl-Coenzyme A Dehydrogenase Associates with a Protein Super-Complex Integrating Multiple Metabolic Pathways

PubMed Central

Narayan, Srinivas B.; Master, Stephen R.; Sireci, Anthony N.; Bierl, Charlene; Stanley, Paige E.; Li, Changhong; Stanley, Charles A.; Bennett, Michael J.

2012-01-01

Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein. PMID:22496890

Topology of subunits of the mammalian cytochrome c oxidase: Relationship to the assembly of the enzyme complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu-Zhong Zhang; Ewart, G.; Capaldi, R.A.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane({sup 35}S)sulfonate and sodium methyl 4-({sup 3}H)formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibodymore » (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C-domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage produce from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.« less

Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene.

PubMed

Li, Anning; Zhang, Yaran; Zhao, Zhidong; Wang, Mingming; Zan, Linsen

2016-01-01

The pyruvate dehydrogenase beta subunit (PDHB) is a subunit of pyruvate dehydrogenase (E1), which catalyzes pyruvate into acetyl-CoA and provides a linkage between the tricarboxylic acid cycle (TCA) and the glycolysis pathway. Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5'-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5'-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5'-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Heung M.; Reed, Jason; Greeley, George H.

Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration inmore » the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase {alpha} subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke.« less

Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene

PubMed Central

Li, Anning; Zhang, Yaran; Zhao, Zhidong; Wang, Mingming; Zan, Linsen

2016-01-01

The pyruvate dehydrogenase beta subunit (PDHB) is a subunit of pyruvate dehydrogenase (E1), which catalyzes pyruvate into acetyl-CoA and provides a linkage between the tricarboxylic acid cycle (TCA) and the glycolysis pathway. Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5’-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5’-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5’-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle. PMID:27379520