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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)
Get PDFAbstractMultifunctional 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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