Dimethyl fumarate protection against collagen II degradation

Degradation of collagen type II caused by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) is one of the major pathological characteristics of osteoarthritis (OA). Dimethyl fumarate (DMF) is a medication approved by the US Food and Drug Administration (FDA) as an oral multiple sclerosis (MS) therapy. In this study, we found that DMF ameliorated collagen type II degradation by inhibiting the expression of MMP-1, MMP-3, and MMP-13 caused by TNF-α. Mechanistically, DMF attenuated MMPs expression by suppressing JAK/STAT3 pathway. These findings imply that DMF treatment might be a potential therapeutic strategy for chondroprotective therapy.     

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.     

Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole,thiabendazole, and levamisole

Comley John C. W. and Wright Spdenis J. 1981. Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole, thiabendazole, and levamisole. International Journal for Parasitology11: 79–84. Succinate dehydrogenase and fumarate reductase activities from a particulate fraction of A. tetraptera and a soluble extract of A. suum have been determined using spectrophotometric methods. Fumarate reductase activity in A. suum could only be detected anaerobically. Succinate dehydrogenase activity from A. suum was partially characterized and shown to exist in several multimolecular forms (isoenzymes). The in vitro effect of the anthelmintics cambendazole, thiabendazole and levamisole on succinate dehydrogenase and fumarate reductase activity from the above nematodes are described. Significant inhibition of fumarate reductase activity of both nematodes was only achieved using 5 mM levamisole and 1 mM thiabendazole. After in vivo anthelmintic treatment of A. tetraptera only thiabendazole significantly inhibited fumarate reductase. It is suggested that the succinate dehydro-ogenase-fumarate reductase complex in these nematodes is unlikely to be the primary site chemotherapeutic attack for any of the anthelmintics tested.     

Anion transport in rat brain mitochondria: Fumarate uptake via the dicarboxylate carrier

Penetration of fumarate into rat brain mitochondria has been investigated, as required in brain ammoniogenesis. Mitochondria swell in ammonium fumarate and this swelling is increased by both Pi and malate. According to a carrier mediated process, fumarate translocation, which occurs in exchange with intramitochondrial malate or Pi shows saturation characteristics. By photometrically investigating the kinetics of fumarate/malate, fumarate/ Pi and malate/Pi exchanges, different Km values were obtained (10, 22 and 250 M, respectively), whereas no significant difference was found forV _max values (40 nmol NAD(P)⁺ reduced/min×mg protein). This suggests that fumarate and malate share a single carrier to enter mitochondria, namely the dicarboxylate carrier. Both comparison made of theV _max values and inhibiton studies exclude a fumarate translocation via either the tricarboxylate carrier, whose occurrence in brain is here demonstrated, or oxodicarboxylate carrier. Kinetic investigation of the dicarboxylate translocator shows the existence of thiol group/s and metal ion/s at or near the substrate binding sites. The experimental findings are discussed in the light of fumarate uptake in vivo in brain ammoniogenesis.Abbreviations AD.SUCC adenylsuccinate - ASP aspartate - BTA 1,2,3,-benzenetricarboxylate - CITR citrate - D-NAD deamino-NAD - PUM fumarate - GABA -aminobutyrate - GAP glyceraldehyde-3-phosphate - GAP-DH glyceraldheyde-3-phosphate dehydrogenase - GHBA -hydroxybutyrate - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - OAA oxaloacetate - OG oxoglutarate - PEP phosphoenolpyruvate - 3-PG glycerate-3-phosphate - 3-PGP glycerate-1,3-diphosphate - PYR pyruvate - RBM rat brain mitochondria - RHM rat heart mitochondria - RKM rat kidney mitochondria - RLM rat liver mitochondria - SSA succinic semialdehyde     

Redox potentials and kinetic properties of fumarate reductase complex from Vibrio succinogenes

The isolated menaquinol: fumarate oxidoreductase (fumarate reductase complex) from Vibrio succinogenes was investigated with respect to the redox potentials and the kinetic response of the prosthetic groups. The following results were obtained. (1) The redox state of the components was measured as a function of the redox potential established by the fumarate/succinate couple, after freezing of the samples (173 K). From these measurements, the midpoint potential of the [2Fe-2S] cluster (−59 mV), the [4Fe-4S] cluster (−24 mV) and the flavin/flavosemiquinone couple (about −20 mV) was obtained. (2) Potentiometric titration of the enzyme in the presence of electron-mediating chemicals gave, after freezing, apparent midpoint potentials that were 30–100 mV more negative than those found with the fumarate/succinate couple. (3) The rate constants of reduction of the components on the addition of succinate or 2,3-dimethyl-1,4-naphthoquinol were as great as or greater than the corresponding turnover numbers of the enzyme in quinone reduction by succinate or fumarate reduction by the quinol. In the oxidation of the reduced enzyme by fumarate, cytochrome b oxidation was about as fast as the corresponding turnover number of quinol oxidation by fumarate, while the [2Fe-2S] and half of the [4Fe-4S] cluster responded more than 2-times slower. The rate constant of the other half of the 4-Fe cluster was one order of magnitude smaller than the turnover number.     

Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1-methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1-methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron–sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.     

Tissue specificity of metabolism in the bivalve mollusc <Emphasis Type="Italic">Anadara inaequivalvis</Emphasis> Br. under conditions of experimental anoxia

Peculiarities of the course of metabolic processes in tissues of the bivalve mollusc Anadara inaequivalvis Br. were studied under conditions of experimental anoxia. In the absence of oxygen, in gill and foot the protein catabolism processes were found to be enhanced; this led to a decrease of the protein content and to an increase of the free amino acid and urea levels. Predominantly hydrolyzed were low molecular peptides, which was indicated by a decrease of the cathepsin D activity on the background of a rise of the γ-glutamyltranspeptidase activity. Anoxia was accompanied by enhancement of the succinate thiokinase and fumarate reductase reactions controlled by alanine and aspartate aminotransferases. This prevented accumulation of toxic lactate in tissues and allowed obtaining an additional macroerg resource. Metabolic processes in the mollusc hepatopancreas were oriented to production of amino acids.     

The role of electrostatics in proton-conducting membrane protein complexes

Electrostatic interactions play a key role in the coupling of electron and proton transfer in membrane protein complexes during the conversion of the energy stored in sunlight or reduced substrates into biochemical energy via a transmembrane electrochemical proton potential. Principles of charge stabilization within membrane proteins are reviewed and discussed for photosynthetic reaction centers, cytochrome c oxidases, and diheme-containing quinol:fumarate reductases. The impact of X-ray structure-based electrostatic calculations on the functional interpretation of these structural coordinates, on providing new explanations for experimental observations, and for the design of more focused additional experiments is illustrated by a number of key examples.     

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 325–332 DOI: 10.1038/sj/jim/7000250 Received 01 October 2001/ Accepted in revised form 12 March 2002     

Enhancement of 1,3-propanediol Production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> with Fumarate Addition

Addition of 5 mm fumarate to cultures of Klebsiella pneumoniae enhanced the rate of glycerol consumption and the production of 1,3-propanediol (PDO). Compared to the control, the activity of glycerol dehydrogenase increased by 35, 33 and 46%, the activity of glycerol dehydratase increased by 160, 210 and 115%, and the activity of 1,3-propanediol oxidoreductase increased by 25, 39 and 85% when, respectively, 5, 15 and 25 mm fumarate were provided. At the same time, the ratio of NAD⁺ to NADH decreased by 20, 23 and 29%. Using a 5 l bioreactor with 5 mM fumarate addition, the specific rate of glycerol consumption and the productivity of PDO was 30 mmol/l h and 17 mmol/l h, respectively, both increased by 35% over the control. Revisions requested 15 July 2005; Revisions received 30 August 2005