http://www.biochemj.org Biochemical Journal RSS feed -- BJ Metabolism Immediate Publications 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/BJ_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110765 2+ (Ca²⁺_cyt) (S_0.5 = 225 ± 22 nM) through its effects on aralar. We now also demonstrate that the a-glycerophosphate-dependent respiration is controlled by Ca²⁺_cyt (S_0.5 = 60 ± 10 nM). At higher Ca²⁺_cyt (< 600 nM), BM accumulate Ca²⁺ which enhances the rate of action of intramitochondrial dehydrogenases. The highest Ca²⁺-induced increments of state 3 respiration decrease with substrate in the order glutamate < a-ketoglutarate < isocitrate < a-glycerophosphate < pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca²⁺_cyt, we show that the formation of pyruvate is tightly controlled by Ca²⁺_cyt. Through its common substrate couple NADH/NAD⁺, the formation of pyruvate by lactate dehydrogenase (LDH) is linked to the malate–aspartate shuttle (MAS) with aralar as a central component. A rise of Ca²⁺_cyt in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to five-fold enhancement of OXPHOS rates that is due to an increased substrate supply, acting in a manner similar to a “gas pedal”. In contrast, mitochondrial Ca²⁺ (Ca²⁺_mit) regulates the oxidation rates of substrates which are present within mitochondrial matrix. We postulate that Ca²⁺_cyt is a key factor in adjusting the mitochondrial energisation to the requirements of intact neurons.]]> F N Gellerich, Z Gizatullina, S Trumbeckaite, B Korzeniewski, T Gaynutdinov, E Seppet, S Vielhaber, H Heinze, F Striggow 2012-02-01T16:25:47Z doi:10.1042/BJ20110765 Portland Press Limited 2012-02-01 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112124 A R Cameron, K Wallace, L Logie, A R Prescott, T G Unterman, J Harthill, G Rena 2012-01-16T14:43:21Z doi:10.1042/BJ20112124 Portland Press Limited 2012-01-16 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111478 Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyze CBS reactions, as demonstrated by analysis of truncated variants and active site-mutant proteins. In contrast to other metazoan enzymes, CBS-1 lacks the heme and the regulatory Bateman domain essential for activation by S-adenosylmethionine and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. This paper provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode cystathionine beta-synthases possess a structural feature that is unique among CBS protein.]]> R Vozdek, A Hnizda, J Krijt, M Kostrouchova, V Kozich 2012-01-13T12:14:15Z doi:10.1042/BJ20111478 Portland Press Limited 2012-01-13 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111057 In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulates both the K_M and the V_M of PARP-1. In addition, we showed that upon treatment by lithium, a very potent inhibitor of the enzyme responsible of pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.]]> E Toledano, V Ogryzko, A Danchin, D Ladant, U Mechold 2012-01-12T14:34:48Z doi:10.1042/BJ20111057 Portland Press Limited 2012-01-12 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111881 i) is present, and that inhibition is lost when P_i is substituted with arsenate (As_i) or vanadate (V_i). To test this, liver mitochondria were isolated from wildtype and CypD-deficient (Ppif^-/-) mice and then incubated in buffer containing P_i, As_i, or V_i. MPT was induced under both energized and de-energized conditions by addition of Ca²⁺, and the resultant mitochondrial swelling measured spectrophotometrically. For pharmacological inhibition of CypD, wildtype mitochondria were pre-incubated with cyclosporine-A (CsA) prior to the addition of Ca²⁺. In energized and de-energized mitochondria, Ca²⁺ induced MPT regardless of the anion present, although the magnitude differed between P_i, As_i, and V_i. However, in all cases, pretreatment with CsA significantly inhibited MPT. Moreover, these effects were independent of mouse strain, organ type, and rodent species. Similarly, attenuation of Ca²⁺-induced MPT in the Ppif^-/- mitochondria was still observed irrespective of whether P_i, As_i, or V_i was present. We conclude that the pharmacological and genetic inhibition of CypD is still able to attenuate MPT even in the absence of P_i.]]> A M McGee, C P Baines 2012-01-11T16:38:05Z doi:10.1042/BJ20111881 Portland Press Limited 2012-01-11 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112026 L Bultot, B Guigas, A Von Wilamowitz-Moellendorff, L Maisin, D Vertommen, N Hussain, M Beullens, J J Guinovart, M Foretz, B Viollet, K Sakamoto, L Hue, M H Rider 2012-01-11T12:10:57Z doi:10.1042/BJ20112026 Portland Press Limited 2012-01-11 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111748 FGF21 gene as a target gene for ATF4 and we have localized two conserved ATF4 binding sequences in the 5’ regulatory region of the human FGF21 gene, which are responsible for the ATF4-dependent transcriptional activation of this gene. These results add FGF21 gene induction to the transcriptional program initiated by increased levels of ATF4 and offer a new mechanism for the induction of the FGF21 gene expression under nutrient deprivation.]]> A De Sousa-Coelho, P F. Marrero, D Haro 2012-01-11T12:04:15Z doi:10.1042/BJ20111748 Portland Press Limited 2012-01-11 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111509 ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol monooxygenase.]]> K Watschinger, J E Fuchs, V Yarov-Yarovoy, M A Keller, G Golderer, A Hermetter, G Werner-Felmayer, N Hulo, E R Werner 2012-01-06T10:56:42Z doi:10.1042/BJ20111509 Portland Press Limited 2012-01-06 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111530 2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. Here we identified several autophosphorylation sites including Thr-348, Thr-353, Ser-366 and Ser-445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser-78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser-78, Thr-348 and Ser-366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr-348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK 293 cells, but not after transfection with a kinase-inactive construct confirming that it is indeed a site of autophosphorylation. Thr-348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other a-kinases is also autophosphorylated and contributes to the activation of these enzymes (Crawley et al., J. Biol. Chem. 2011, 286, 2607-2616). Ser-366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is not only phosphorylated autocatalytically but also in trans by other kinases.]]> S Pyr Dit Ruys, X Wang, E M Smith, G Herinckx, N Hussain, M H Rider, D Vertommen, C Gregory Proud 2012-01-04T15:16:12Z doi:10.1042/BJ20111530 Portland Press Limited 2012-01-04 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111280 C Arden, S J Tudhope, J L Petrie, Z H Al-Oanzi, K S Cullen, A J. Lange, H C Towle, L Agius 2012-01-03T16:07:36Z doi:10.1042/BJ20111280 Portland Press Limited 2012-01-03 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111648 ca. 2-fold. We conclude that dicarbonyl stress is countered by up-regulation of glyoxalase-1 in the Nrf2 stress responsive system, protecting protein and DNA from increased damage and preserving cell function.]]> M Xue, N Rabbani, H Momiji, P Imbasi, M Anwar, N Kitteringham, K Park, T Souma, T MORIGUCHI, M Yamamoto, P J. Thornalley 2011-12-22T10:54:17Z doi:10.1042/BJ20111648 Portland Press Limited 2011-12-22 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111420 + are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In this work we have functionally identified and characterized SLC25A17, which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Herein, recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN, AMP and to a lesser extent of NAD⁺, adenosine 3’,5’-diphosphate (PAP) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and inhibited by pyridoxal-5’-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD⁺ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. This is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.]]> G Agrimi, A Russo, P Scarcia, F Palmieri 2011-12-21T12:58:16Z doi:10.1042/BJ20111420 Portland Press Limited 2011-12-21 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111849 Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labeling studies using [methyl-²H₃]methionine and substrate/product-specificities of the cloned sterol C24-methyl transferase (24-SMT) and sterol C14-demethylase (14-SDM) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ergosta-5,7,25(27)-trien-3β-ol (ETO), 24-dimethyl ergosta-5,7,25(27)-trienol (DTO) and ergosta-5,7,22(23)-trienol (ergosterol). To assess the possible carbon sources of ergosterol biosynthesis, specifically ¹³C-labeled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the ¹³C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternate precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the ¹³C-labeling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected in the progressive increase in ¹³C-ergosterol production (control < [2-¹³C]leucine < [2-¹³C]acetate < [1-¹³C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol yielding distinct sterol profiles in relation to cell demands on growth.]]> C Robert Nes, U K Singha, J Liu, K Ganapathy, F Villalta, M R. Waterman, G I Lepesheva, M Chaudhuri, W David Nes 2011-12-19T12:18:58Z doi:10.1042/BJ20111849 Portland Press Limited 2011-12-19 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112037 in vitro and in vivo, and this effect is dependent on intact signaling via phosphatidylinositol 3-kinase (PI3K), but not protein kinase B (PKB). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. Firstly, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenoviral-mediated over-expression of wild type (WT) IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signaling was not potentiated in mouse embryo fibroblasts lacking IKKβ (Ikkβ^-/- MEF). Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our data that IKKβ, while activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin-signaling cascade.]]> G I Lancaster, B Skiba, C Yang, H T Nicholls, K G Langley, M Chan, C R Bruce, G W Rewcastle, P R Shepherd, M Karin, M A. Febbraio 2011-12-14T12:16:28Z doi:10.1042/BJ20112037 Portland Press Limited 2011-12-14 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111796 Synechocystis sp. PCC6803 and report here on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an E. coli mutant with a defect in ubiquinone biosynthesis as well as by in vitro assays using the recombinant as well as the native enzyme. While Slr0926 was highly specific for the prenyl acceptor substrate, 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate (SPP) but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferase catalysing prenylation in the course of ubiquinone biosynthesis.]]> R Sadre, C Pfaff, S Buchkremer 2011-12-14T11:13:05Z doi:10.1042/BJ20111796 Portland Press Limited 2011-12-14 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111166 S Bhatia, B Knoch, J Wong, W Scott Kim, P L. Else, A J Oakley, B Garner 2011-12-12T15:05:50Z doi:10.1042/BJ20111166 Portland Press Limited 2011-12-12 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111311 Arabidopsis. The BCD1 gene is induced by excessive iron but repressed by iron deficiency. It is also induced by cellular and tissue damages occurring under osmotic stress. The activation-tagged mutant bcd1-1D exhibits leaf chlorosis, typical symptom of iron deficiency. The chlorotic lesion of the mutant was partially recovered by iron feeding. Whereas the bcd1-1D mutant accumulated a lower amount of iron, the iron level was elevated in the knockout mutant bcd1-1. The BCD1 protein is localized to the Golgi complex. We propose that the BCD1 transporter plays a role in sustaining iron homeostasis by reallocating excess iron released from stress-induced cellular damages.]]> P Seo, J Park, M Park, Y Kim, S Kim, J Jung, C Park 2011-12-08T11:38:33Z doi:10.1042/BJ20111311 Portland Press Limited 2011-12-08 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111618 Lactococcus lactis cannot synthesize heme, but when supplied with heme, expresses a cytochrome bd oxidase. Aside of the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in heme metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is know for hemW. HemW-like proteins occur in bacteria, plants, and animals, and are usually annotated as coproporphyrinogen III dehydrogenases. However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of coproporphyrinogen III dehydrogenase activity in vivo and in vitro. Recombinantly produced, purified HemW contained an iron-sulfur cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound heme b in vitro, with a stoichiometry of one heme per monomer and a K_D of 8 µM. In vivo, HemW occurred as a heme free, cytosolic form, as well as a heme-containing, membrane-associated form. Addition of L. lactis membranes to heme-containing HemW triggered the release of heme from HemW in vitro. Based these findings, we propose a role of HemW in heme trafficking. HemW-like proteins form a distinct phylogenetic clade which has not previously been recognized.]]> H K Abicht, J Martinez, G Layer, D Jahn, M Solioz 2011-12-05T14:10:46Z doi:10.1042/BJ20111618 Portland Press Limited 2011-12-05 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111418 2), a key modulator of glycolysis and gluconeogenesis. PFKFB3 gene is extensively involved in cell proliferation due to its key role in carbohydrate metabolism. Here we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or Norgestrel) leads to a rapid increase in Fru-2,6-P₂ concentration. Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a progesterone regulatory element (PRE) to which progesterone receptor binds and thus transactivates PFKFB3 gene transcription. Progesterone Receptor (PR) expression, in the PR-negative cell line (MDA-MB-231), induces endogen PFKFB3 expression in response to Norgestrel. Direct Binding of PR to the PRE box (-3490 nt) was confirmed by ChIP experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK-RSK pathway leading to phosphorylation of PFKFB3 on Ser 461 is followed by activation of mRNA transcription via cis-acting sequences on PFKFB3 promoter.]]> L Novellasdemunt, M Obach, L Millán-Ariño, A Manzano, F Ventura, J Rosa, A Jordan, A Navarro-Sabate, R Bartrons 2011-11-24T14:08:00Z doi:10.1042/BJ20111418 Portland Press Limited 2011-11-24 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111005 Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic δ-aminolevulinate synthase 1 (ALAS-1), first and rate-limiting enzyme of haem pathway. Porphyria acute attacks are usually treated with glucose administration, its effect is apparently related to its ability of inhibiting ALAS-1 by modulating insulin plasma levels. It was shown that insulin blunts hepatocytes ALAS-1 induction, by disrupting the interaction of the Forkhead box O1 (FOXO1) and the proliferator-activated receptor γ coactivator 1α (PGC-1α). We evaluated the expression of ALAS-1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadate, on the enzyme regulation to evaluate its potential for the treatment of porphyria acute attacks. Both ALAS-1 mRNA and protein content were induced in diabetic animals, accompanied by decreased Akt phosphorylation and increased nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex. Vanadate reversed ALAS-1 induction with a concomitant PI3K/Akt pathway activation and subsequent reduction of nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. These finding support that the FOXO1-PGC-1α complex is involved in the control of ALAS-1 expression and further suggest that a vanadate-based therapy could be beneficial for the treatment of porphyria acute attacks. ]]> L Maria Oliveri, C Davio, A María del Cármen Batlle, E Noemi Gerez 2011-11-10T09:13:02Z doi:10.1042/BJ20111005 Portland Press Limited 2011-11-10 BJ Metabolism