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The Compartmentalization of Folate Metabolism in Mammalian Cells

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The Compartmentalization of Folate Metabolism in Mammalian Cells The Compartmentalization of Folate Metabolism in Mammalian Cells by Harshila Patel A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Doctor of Philosophy Department of Biochemistry McGili University Montreal, Quebec, Canada © by Harshila Patel June, 2004 Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 0-494-06334-3 Our file Notre référence ISBN: 0-494-06334-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada This thesis is dedicated to my wonderfully supportive parents, Ashvin and Jyoti ABSTRACT Folate metabolism is compartmentalized between the cytoplasm and the mitochondria of mammalian cells. Certain folate-dependent enzymes are present in both of these compartments, such as methylenetetrahydrofolate dehydrogenases, which are required to interconvert one-carbon substituted tetrahydrofolates. In the cytoplasm, there is a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase (DCS). Its mitochondrial counterpart is a bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC), expressed during embryogenesis and in immortalized cells. A comparison of the 3' untranslated region of the NMDMC cDNA with the synthetase domain of the DCS cDNA and gene among different species have revealed significant regions of homology. This suggests that the mammalian mitochondrial NAD-dependent OC evolved from an NADP-dependent DCS precursor through a change in cofactor specificity of the dehydrogenase from NADP to NAD. Although the folate pathways are compartmentalized, the mitochondrial folate pathway makes an important contribution to total cellular folate metabolism. Mouse fibroblasts that have a completely inactivated NMDMC gene are glycine auxotrophs. Furthermore, growth of these Nmdmc-/- cell lines is stimulated by supplementation with formate or hypoxanthine. These cell lines also show enhanced incorporation of radioactivity into DNA from formate as compared to serine, demonstrating that formate is a preferred one-carbon donor for the Nmdmc-/- cell lines. This indicates that NMDMC is required for optimal purine biosynthesis during periods of rapid cellular proliferation su ch as embryogenesis and tumorigenesis. The rescue of these Nmdmc-/- cell lines with NMDMC expression reversed the glycine auxotrophy and the formate to serine incorporation ratio reverted toward the wild type ratio. The rescue of these Nmdmc-/- cell lines with the NAD-dependent monofunctional dehydrogenase activity also reversed the glycine auxotrophy but these cell lines did not grow as weil as the NMDMC-rescued ce Il lines. This indicates that although the cyclohydrolase activity is not required in the mitochondria, the rate of 10- formylTHF production is not optimal in its absence. Furthermore, when these Nmdmc-/- cell lines were rescued with the expression of the NADP-dependent DCS in the mitochondria there was reversai of the glycine auxotrophy as weil, indicating that the NAD cofactor specificity of the mitochondrial methylenetetrahydrofolate dehydrogenase is not absolutely essential to maintain the flux of one-carbon metabolites. RÉSUMÉ Dans les cellules de mammifères, le métabolisme du folate est compartimenté entre le cytoplasme et les mitochondries. Certaines enzymes qui dépendent du folate sont présentes dans ces deux compartiments, comme par exemple, les déshydrogénases de méthylènetétrahydrofolate nécessaire à l'interconversion des tétrahydrofolates porteurs d'unité de carbone. Le cytoplasme contient une enzyme trifonctionnelle, la déshydrogénase de méthylènetétrahydrofolate dépendante du NADP-cyclohydrolase-synthétase (DCS) dont l'équivalent mitochondrial est une enzyme bifonctionnelle: la déshydrogénase de méthylènetétrahydrofolate dépendante du NAD­ cyclohydrolase (NMDMC). Cette dernière est exprimée durant l'embryogenèse et dans les lignées de cellules transformées. Une comparaison de la région 3' non traduite de l'ADNe de NMDMC avec le domaine de synthétase de l'ADNe et du gène de DCS provenant d'espèces différentes a indiqué des régions significativement homologues. Ceci suggère que l'enzyme mitochondriale de mammifères, la OC dépendante du NAD, aurait évoluée à partir d'un précurseur de la DCS dépendante du NADP ayant subi un changement de cofacteur, passant du NADP au NAD. Bien que les voies de signalisation du folate soient compartimentées, la voie mitochondriale apporte une contribution importante au métabolisme cellulaire du folate. Les fibroblastes de souris dont le gène NMDMC est complètement inactivé sont auxotrophes pour la glycine. De plus, la croissance de ces cellules I Nmdmc- - est stimulée par la présence de formate ou d'hypoxanthine. Ces cellules montrent également plus d'incorporation de radioactivité à l'ADN lorsqu'on utilise du formate au lieu de la sérine, ce qui démontre l'utilisation préferentielle du formate en tant que donneur d'unités de carbone pour ces cellules. Les résultats indiquent que la NMDMC est nécessaire à la biosynthèse optimale des purines pendant les périodes de prolifération cellulaire rapide telles I que l'embryogenèse ou la tumorigenèse. Lorsque dans ces cellules Nmdmc- - on restaure la NMDMC par transfection, l'auxotrophie pour la glycine est renversée et le rapport d'incorporation préferentielle du formate par rapport à la sérine revient à la normale. Lorsque l'activité monofonctionnelle de déshydrogénase à I NAD est restaurée dans les cellules Nmdmc- -, l'auxotrophie pour la glycine est renversée, mais les lignées cellulaires ne prolifèrent pas aussi bien que les I cellules Nmdmc- - exprimant de nouveau la NMDMC. Ceci indique que, bien que l'activité de la cyclohydrolase ne soit pas nécessaire dans les mitochondries, le taux de production du 10-formyltétrahydrofolate n'est pas optimal en son absence. De plus, quand on exprime la DCS dépendante du NADP dans les mitochondries I des cellules Nmdmc- - par transfection, l'auxotrophie pour la glycine est de nouveau renversée, indicant que la spécificité pour le NAD de la déshydrogénase de méthylènetétrahydrofolate mitochondriale n'est pas absolument essentielle au maintient du flot des métabolites d'unité de carbone. TABLE OF CONTENTS FOREWORD .......................................................................................................... i ACKNOWLEDGEMENTS ..................................................................................... v PUBLICATION OF THE WORK PRESENTED IN THIS THESIS .......................viii CONTRIBUTIONS TO ORIGINAL KNOWLEDGE .............................................. ix LIST OF FIGURES .............................................................................................. xii LIST OF TABLES ...............................................................................................xiv LIST OF ABBREVIATIONS .................................................................................xv CHAPTER ONE: GENERAL INTRODUCTION ............................................. 1 OISCOVERY OF FOLIC ACIO .......................................................................... 2 FOLIC ACIO ...................................................................................................... 2 FOLATE ABSORPTION .................................................................................... 4 MECHANISMS OF CELLULAR TRANSPORT .................................................. 5 FOLATE BINDING PROTEINS ........................................................................... 6 Folate Receptor ............................................................................................... 6 Reduced Folate Carrier ................................................................................... 9 LOW PH TRANSPORTER
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