76 research outputs found
In Saccharomyces cerevisiae grown in synthetic minimal medium supplemented with non-fermentable carbon sources glutamate is synthesized within mitochondria
No full textIn Saccharomyces cerevisiae the export of 2-oxoglutarate from the mitochondria, catalyzed by Yhm2p, Odc1p and Odc2p or by at least one of these transporters, has recently been shown to be essential for glutamate biosynthesis in glucose-supplemented minimal synthetic (SM) medium without glutamate, because the triple mutant yhm2∆odc1∆odc2∆ displays a growth defect under these conditions. Surprisingly, in this study it was found that yhm2∆odc1∆odc2∆ cells grow like wild-type (WT) cells in the same medium supplemented with non-fermentable carbon sources. Direct transport assays of 2-oxoglutarate/2-oxoglutarate homoexchange activity in mitochondria from WT and yhm2∆odc1∆odc2∆ cells (solubilized and reconstituted into liposomes) showed that the mitochondrial extract from yhm2∆odc1∆odc2∆ was completely inactive at variance with that from WT cells, showing that S. cerevisiae mitochondria do not contain additional proteins capable of catalyzing 2-oxoglutarate transport efficiently besides Yhm2p, Odc1p and Odc2p. Furthermore, quantitative real-time PCR experiments showed that in both WT and yhm2∆odc1∆odc2∆ cells the expression of GDH1 is low on lactate and high on glucose and, vice versa, the expression of GDH3 is high on lactate and low on glucose. These results may be interpreted to indicate that in S. cerevisiae, grown in glucose-supplemented SM medium, glutamate is synthesized by Gdh1p in the cytosol, whereas in lactate-supplemented SM medium glutamate is synthesized by Gdh3p in the mitochondria; therefore, the pathway of ammonia assimilation under fermentative conditions requires export of 2-oxoglutarate from the mitochondria, whereas the alternative pathway under respiratory conditions does not
Role of mitochondrial 2-oxoglutarate transporters in the assimilation of nitrogen in S. cerevisiae.
No full textThe nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three mitochondrial 2-oxoglutarate transporters. Mitochondrial extract from yhm2∆odc1∆odc2∆ cells reconstituted into liposomes showed that 2-oxoglutarate/2-oxoglutarate homo-exchange activity was completely inactive, showing that in S. cerevisiae Yhm2p, Odc1p and Odc2p are the sole 2-oxoglutarate transporters.
Both the odc1Δodc2Δ double knockout and the yhm2Δ mutants showed a growth like that of the wild-type strain on a synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2Δodc1Δodc2Δ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain and by the addition of glutamate, but not glutamine, to the medium.
Surprisingly, it was found that yhm2Δodc1Δodc2Δ cells grew like wild-type cells in the lactate-supplemented SM medium without glutamate. In S. cerevisiae, glutamate is synthesized by glutamate dehydrogenase isoforms (GDH). GDH1 is localized in the cytosol and expressed when cells grow on glucose, while GDH3 is localized in the mitochondria and expressed when cells grow on non-fermentable carbon sources. By quantitative real-time PCR experiments we showed that in both WT and yhm2Δodc1Δodc2Δ cells the expression of cytosolic GDH1 is low on lactate and high on glucose and, vice versa, the expression of mitochondrial GDH3 is high on lactate and low on glucose, suggesting that in lactate-supplemented SM medium glutamate biosynthesis is synthesized in the mitochondria by Gdh3p.
In conclusion, the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of 2-oxoglutarate required in the cytosol for glutamate when cells grew in glucose-supplemented medium
Identification and functional characterization of a novel mitochondrial carrier for citrate and oxoglutarate in Saccharomyces cerevisiae
No full textMitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP(+) and GSH/GSSG ratios in the cytosol of DeltaYHM2 cells as well as an increase in the NADPH/NADP(+) ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the DeltaYHM2 strain and more so by the DeltaYHM2DeltaZWF1 strain upon H(2)O(2) exposure, implying that Yhm2p has an antioxidant function
Structural studies on the enzymatic units of the peroxisomal multifunctional enzyme type 2 (MFE-2)
No full textAbstractMultifunctional enzyme type 2 (MFE-2) is a peroxisomal enzyme participating in the breakdown of fatty acids in eukaryotes. Depending on the organism, MFE-2 is composed of two to four functional units, out of which the two enzymatic ones, 2-enoyl-coenzyme A (CoA) hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase, are found in the all MFE-2s. These units are responsible for the catalysis of the second and third steps of the peroxisomal β-oxidation of various CoA thioesters of fatty acids and fatty acyl derivatives. Their (R)-stereospecificity and ability to accept a broad range of fatty acid CoA esters as substrates, in addition to the fact that they do not share any sequence similarity with the classical mitochondrial counterparts, make the enzymatic units of MFE-2 structurally very interesting. In this study, the three-dimensional structures of the (3R)-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase 2 units were solved by crystallographic methods.The crystal structure of the (3R)-hydroxyacyl-CoA dehydrogenase unit of rat MFE-2 reveals a dimeric enzyme with an α/β short-chain alcohol dehydrogenase/reductase (SDR) fold. A unique feature of (3R)-hydroxyacyl-CoA dehydrogenase, however, is the separate C-terminal domain, which completes the active site cavity of the adjacent monomer and extends the dimeric interactions. The 2-enoyl-CoA hydratase 2 unit is a dimer with a unique two-domain structure proposed to evolve via gene duplication. The fold consists of two side-by-side arranged repeats of the hot-dog fold motifs, thus being highly reminiscent of the tertiary structures of the (R)-specific 2-enoyl-CoA hydratase of the polyhydroxyalkanoate synthesis pathway and the β-hydroxydecanoyl thiol ester dehydrase of fatty acid synthesis type II, both from prokaryotic sources. The importance of the N-domain in the binding of bulky substrates was shown by the enzyme-product complex structure, which also indicates the active site. For the first time, it was shown that the eukaryotic hydratase 2 uses an Asp/His catalytic dyad in catalysis. Moreover, a novel catalytic mechanism was proposed for (R)-specific hydration/dehydration.The solved structures also provide a molecular basis for understanding the effects of the patient mutations of MFE-2. They also allow disussion of the possible organisation of the three units in full-length MFE-2 of mammals.Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on November 5th, 2004, at 12 noon.Abstract
Multifunctional enzyme type 2 (MFE-2) is a peroxisomal enzyme participating in the breakdown of fatty acids in eukaryotes. Depending on the organism, MFE-2 is composed of two to four functional units, out of which the two enzymatic ones, 2-enoyl-coenzyme A (CoA) hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase, are found in the all MFE-2s. These units are responsible for the catalysis of the second and third steps of the peroxisomal β-oxidation of various CoA thioesters of fatty acids and fatty acyl derivatives. Their (R)-stereospecificity and ability to accept a broad range of fatty acid CoA esters as substrates, in addition to the fact that they do not share any sequence similarity with the classical mitochondrial counterparts, make the enzymatic units of MFE-2 structurally very interesting. In this study, the three-dimensional structures of the (3R)-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase 2 units were solved by crystallographic methods.
The crystal structure of the (3R)-hydroxyacyl-CoA dehydrogenase unit of rat MFE-2 reveals a dimeric enzyme with an α/β short-chain alcohol dehydrogenase/reductase (SDR) fold. A unique feature of (3R)-hydroxyacyl-CoA dehydrogenase, however, is the separate C-terminal domain, which completes the active site cavity of the adjacent monomer and extends the dimeric interactions. The 2-enoyl-CoA hydratase 2 unit is a dimer with a unique two-domain structure proposed to evolve via gene duplication. The fold consists of two side-by-side arranged repeats of the hot-dog fold motifs, thus being highly reminiscent of the tertiary structures of the (R)-specific 2-enoyl-CoA hydratase of the polyhydroxyalkanoate synthesis pathway and the β-hydroxydecanoyl thiol ester dehydrase of fatty acid synthesis type II, both from prokaryotic sources. The importance of the N-domain in the binding of bulky substrates was shown by the enzyme-product complex structure, which also indicates the active site. For the first time, it was shown that the eukaryotic hydratase 2 uses an Asp/His catalytic dyad in catalysis. Moreover, a novel catalytic mechanism was proposed for (R)-specific hydration/dehydration.
The solved structures also provide a molecular basis for understanding the effects of the patient mutations of MFE-2. They also allow disussion of the possible organisation of the three units in full-length MFE-2 of mammals
N-Glycan Modification of Recombinant Factor IX -
No full textProtein- und Peptidtherapeutika sind aus der modernen Medizin heutzutage nicht mehr wegzudenken. Obwohl diese Medikamentenklasse viele Vorteile gegenüber konventionellen Arzneimitteln hat, gibt es auch einige Unzulänglichkeiten, vor allem die schlechte Pharmakokinetik. Eine sehr gute Möglichkeit um diese Schwäche zu kompensieren besteht darin ein Polymer kovalent an den Wirkstoff zu koppeln um dadurch dessen positive Eigenschaften auf das Präparat zu übertragen.Das Ziel dieser Arbeit war es eine neuartige Kopplungsstrategie für die chemische Modifizierung von rekombinanten Blutgerinnungsfaktoren zu entwickeln, im Speziellen Faktor IX gekoppelt mit Polysialinsäure. Durch solch ein chemisch modifiziertes Faktor IX-Derivat kann die Lebensqualität von Hämophilie B Patienten erheblich verbessert werden, weil dadurch eine effektive prophylaktische Therapie ermöglicht wird, wie sie zurzeit nicht erhältlich ist.Das Ziel einer solchen Modifikation von Faktor IX sind die beiden im Aktivierungspeptid gelegenen N-Glykane. Diese Herangehensweise gestattet ein ,Drug-Design' bei dem das chemisch modifizierte Proenzym im Blutkreislauf zirkuliert und nach der Aktivierung das native unmodifizierte Enzym freisetzt. Während der Entwicklung eines solchen Hämophilie Präparates wurden verschiedene Arten der chemischen Modifizierung, sowie verschieden Reaktionsanordnungen und auch verschiedene chromatographische Aufreinigungsmethoden evaluiert.Außerdem wurde die Polysialinsäure, welche zur Faktor IX-Modifikation verwendet wurde, in einer abgewandelten reduktiven Aminierung zu einem neuartigen aminooxy-funktionalisierten Polymer umgesetzt. Des Weiteren wurde der Einfluss eines nukleophilen Katalysators für die Konjugationsreaktion untersucht. Diese wissenschaftliche Arbeit zeigt die Realisierbarkeit der Herstellung eines Faktor IX-Polysialinsäure Präparats in Bezug auf zwei entscheidende Punkte. Zuallererst konnten durch dieses Verfahren Faktor IX-Derivate hergestellt werden, welche spezifische Aktivitäten von bis zu 63 % behielten und verbesserte Pharmakokinetik in verschieden Tiermodellen zeigten. Des Weiteren ermöglichte eine Aufreinigungsmethode, bestehend aus einem Anionentauscherschritt und einer Hydrophoben-Interaktionschromatographie, Proteinausbeuten von bis zu 73 % und Aktivitätsausbeuten von bis zu 48 %, was die industrielle Herstellung eines solchen Präparates überhaupt erst interessant macht.Protein and peptide therapeutics are indispensible in modern medicine today. Despite their great therapeutic benefits this class of drugs suffers from several shortcomings, most importantly their poor pharmacokinetics. One of the most elaborate ways to compensate this weakness is the conjugation of a polymeric moiety like polyethylene glycol, dextran or polysialic acid, to the biopharmaceutical drug, thereby endowing it with all the favorable properties of the polymer. The objective of this work was to develop a novel conjugation strategy for the chemical modification of recombinant blood coagulation factors, in particular factor IX conjugated to polysialic acid. Such a modified factor IX derivative has the ability to substantially improve the quality of life of people suffering from hemophilia B by providing an effective prophylactic therapy, which is not available by now. The target of the modification within factor IX were the two N-glycans located on the activation peptide. This conjugation approach allows a drug design whereat the modified pro-enzyme is circulating in the blood stream and releases the active un-modified enzyme upon activation. During the development of such a hemophilia B therapeutic different conjugation chemistries (reductive amination, hydrazone- and oxime formation) targeted to oxidized N-glycans, various conjugation designs (sequential- and simultaneous reaction approach) and also diverse chromatographic purification procedures were evaluated. Additionally the polymer that has been used for the modification (polysialic acid oxidized at the non-reducing end) was derivatized in a modified reductive amination reaction creating a novel aminooxy-functionalized polymer. Furthermore the necessity of nucleophilic catalysts for the conjugation reaction was assessed. The present work demonstrates the feasibility for the preparation of a factor IX-polysialic acid conjugate with regard to two vital aspects. First and foremost, through the developed conjugation approach, that is a concomitant oxidation of factor IX and conjugation to polysialic acid in the presence of m-toluidine as innoxious nucleophilic catalyst, a factor IX derivative was prepared that retained up to 63 % of its specific activity and showed enhanced pharmacokinetics in various animal models. Secondly, the corresponding purification process, that is a 2-column method employing an anion exchange and a hydrophobic interaction chromatography step, yielded in product recoveries of up to 73 % of protein and up to 48 % of activity units, making industrial manufacturing feasible
Farnesylation of Pex19p is required for its structural integrity and function in Peroxisome Biogenesis
No full textThe conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions
The biochemistry of oleate induction: Transcriptional upregulation and peroxisome proliferation
No full textAbstractUnicellular organisms such as yeast constantly monitor their environment and respond to nutritional cues. Rapid adaptation to ambient changes may include modification and degradation of proteins; alterations in mRNA stability; and differential rates of translation. However, for a more prolonged response, changes are initiated in the expression of genes involved in the utilization of energy sources whose availability constantly fluctuates. For example, in the presence of oleic acid as a sole carbon source, yeast cells induce the expression of a discrete set of enzymes for fatty acid β-oxidation as well as proteins involved in the expansion of the peroxisomal compartment containing this process. In this review chapter, we discuss the factors regulating oleate induction in Saccharomyces cerevisiae, and we also deal with peroxisome proliferation in other organisms, briefly mentioning fatty acid-independent signals that can trigger this process
The ins and outs of peroxisomes: Co-ordination of membrane transport and peroxisomal metabolism
No full textAbstractPeroxisomes perform a range of metabolic functions which require the movement of substrates, co-substrates, cofactors and metabolites across the peroxisomal membrane. In this review, we discuss the evidence for and against specific transport systems involved in peroxisomal metabolism and how these operate to co-ordinate biochemical reactions within the peroxisome with those in other compartments of the cell
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