PYRUVATE DEHYDROGENASE COMPLEX - CORRELATION BETWEEN STRUCTURE AND FUNCTION AGNIESZKA SEYDA A thesis submitted in conforrnity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochernistry University of Toronto @Copyright by Agnieszka Seyda National Library Bibliothèque nationale ($1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Sewices selvices bibliographiques 395 WeUington Street 395. rue Wellington Ottawa ON Ki A ON4 Ottawa ON K 1A ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive pemettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel reproduire, prêter, distribuer ou copies of ths thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de rnicrofiche/film, de reproduction sur papier ou sur foxmat électronique. The author retains ownership of the L'auteur conserve la propriété ch copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othewise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ACKNOWLEDGEMENTS I wish to express my thanks and gratitude to Dr. Brian Robinson for his support, assistance and most of al1 patience during the entire course of this degree. His stimulating discussions always served to heighten my enthusiasm to cary out the research and his knowledge made me realize that there will never be the end to my education. I would like to thank the rnembers of my Ph.D. committee, Dr. John Callahan and Dr. James Ellis for al1 their encouragement. support and constructive criticism. 1 would also like to thank my [ab mates. some of who deserve a special mention. Dr. Mingfu Ling and Dr. Sandeep Raha were especially critical to my science education. never too busy to help out as well as talk about science. Finally, 1 would like to thank my family. My parents have been an endless source of support (both emotionai and financial). They always told me not to be afraid to take risk in life and remove limits from my dreams. I would also like to thank my husband Roberto who taught me that it is not important what you do but why you do it. PYRUVATE DEHYDROGENASE COMPLEX - CORRELATION BETWEEN STRUCTURE AND FUNCTION Doctor of Philosophy, 2001 Agnieszka Seyda Department of Biochemistry, University of Toronto ABSTRACT Human PDH complex deficiency is an extremely heterogenous disease in its presentation. Very little correlation between clinical course and molecular characteristics has becn established SJ far. In order to more accurately define the structure of the complex. mutationai analyses were perfomed on its two components, PDH-E,aand protein X. A series of PDH- E,a deletion mutants was construcied and expressed in a ce11 line with zero PDH complex activity due to a nul1 E,a allele (the no ce11 line). Sequential deletion of the C-terminus by 1,2,3 and 4 amino acids resulted in PDH complex activities of 100, 60. 30 and 14% compared to wild-type Ela expressed in PDH complex deficient cells. In addition the somatic and the testis-specific PDH-E,a human isoforms were determined to be biochemically equivalent. Furthermore. a series of PDH-Ela histidine mutants (H63.H84. H92 and H263) was constructed and expressed in the d' cell line. H63 and H263 were shown to be essential for the PDH complex activity. while Hg2 was implicated in the PDH-Ela,& heterotetramer formation and/or stability. A novel PDH-Ela mutation R 14 1 L was characterized in a male patient and it was found in about 75% of the studied DNA derived fom various tissues (fibroblasts, liver and muscle). This scenario would indicate a case of E,a somatic mosaicism. The R141L mutation is a severe one and it must have occurred in iii one of the E,a alleles during early embryogenesis. To gain further insight into the nature and function of the domains of the human protein X. two artificially created variants. K37E and S422H as well as the wild-type were expressed in SV40-immortalized protein X-deficient and EL-deficient human skin fibroblasts. The former mutant does not carry the lipoic acid moiety; the latter mutant was designed to investigate the possibility that protein X could exhibit an intrinsic rransacetylase activity. None of the expressed protein X variants had an effect on the specific activity of the PDH complex. suggesting that the human protein X plays a purely structural role. A novel syndrome affecting multiple mitochondrial functions was described and localized by microcell-mediated transfer to chromosornr 2p 14-2p 13. TABLE OF CONTENTS ACKNOWLEDGEMENTS ii ABSTRACT iii TABLE OF CONTENTS v LIST OF FIGURES viii LIST OF TABLES X ABBREVIATIONS xi NOTICE xiii CHAPTER 1 INTRODUCTION What are mitochondria 2-0x0 acid dehydrogenases - overview The pyruvate dehydrogenase complex El Thiamin metabolism E,a - the testis-specific isoform E, and protein X ~lpicacid metabolisrn E3 Regulation of the PDH complex Extracellular regulation of the PDH complex Import into rnitochondria Commercial uses of 2-0x0 acid dehydrogenases Mutational studies of the PDH-E,a Expression systems The metabolon Objectives of the thesis CHAPTER 2 SEQUENTIAL DELETION OF C-TERMINAL AMINO ACIDS OF THE Ela COMPONENTS OF THE PDH COMPLEX LEADS TO WDUCED STEADY-STA'E LEVELS OF FUNCTIONAL Ela$, TETRAMERS: IMPLICATIONS FOR PATIENTS WITH PDH DEFICIENCY 2.1 Introduction 2.2 Materials and Methods 2.3 Results 2.4 Discussion 2.5 Acknowledgements CHAPTER 3 FUWCTIONAL EXPRESSION OF FOUR PDH-E,a RECOMBINANT HISTIDINE iMUTANTS IN A HUMAN FIBROBLAST CELL LINE WITH ZERO ENDOGENOUS PDH COMPLEX ACTIVITY 3.1 Introduction 3.2 Materials and Methods 3.3 Results 3.4 Discussion 3.5 Acknowledgements CHAPTER 4 EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF THE HUMAN PROTEIN X VARIANTS IN SV40-IMMORTALIZED PROTEIN X-DEFICIENT AND E2-DEFICIENT HUMAN SKIN FIBROBLASTS 4.1 Introduction 4.2 Materials and Methods 4.3 Results and Discussion 4.4 Acknowledgements CHAPTER 5 A CASE OF PDH-Ela MOSAICISM IN A MALE PATIENT WITH SEVERE METABOLIC LACTIC ACIDOSIS 5.1 Introduction 5.2 Materials and Methods 5.3 Results 5 -4 Discussion 5.5 Acknowledgements CHAPTER 6 A NOVEL SYNDROME AFFECTING MULTIPLE bIITOCHONDRIAL FUNCTIONS, LOCATED BY MICROCELL-MEDIATED TRANSFER TO CHROMOSOME 2p14-2p13 6.1 Introduction 6.2 Case report 6.3 Materials and Methods 6.4 Results 6.5 Discussion CHAPTER 7 DISCUSSION AND FUTURE DIRECTIONS 7.1 Cornparison of PDHC with BCKDHC and OGDHC 14 1 7.2 Cornparison of the E, forms 142 7.3 Protein X 153 7.4 Multiple rnitochondrial enzyme disorders 156 7.5 Future directions 157 REFERENCES 159 vii LIST OF FIGURES CHAPTER 1 Schematic drawing of a rnitochondrion Position of the pyruvate dehydrogenase complex (PDHC) within cellular metabolism (A), Reaction sequence of PDHC (B) The chernical stmcture of thiamin diphosphate in its ylid form (A). proposed mechanism of ThDP-assisted decarboxylation of pyruvate by human El (B) Schematic outline of thiamin rnetabolism in yeast The domain structure of the acety! transferase core enzyme From E. di,mammals and yeast (A) and the mammalian/yeast protein X (B) The structure of the lipoyl domain from A. vinelandii The last step of lipoic acid synthesis carried out by lipoate synthase Schematic drawing of the active site of A. vinelandii E3 (A). proposed reaction of E3 (B) The control of phosphorylation-dephosphorylation of py nivate dehydrogenase by various effectors Schematic diagram showing mitoc hondrial cal' uptake sites in juxtaposition to the IP3-sensitive ca2+release channrl Protein import pathways into mitochondria Alignrnent of 6 Eiasequences (A); Alignment of 6 El sequences CHAPTER 2 2.1 Westem blot analysis of cultured skin fibroblasts (A), and lymphoblasts (B) derived from patients with ElaC-terminal deietions 2.2 Sequencing of plasmids containing PCR products of PDH-Ela cDNA derived from patients with E!a C-terminal de letions 2.3 Western blot analysis of the no celi line as well as the parental and control ceil lines 2.4 Westem blot analysis of plasmid expression of the wild-type and variant PDH-Ela 2.5 Quantitative RT-PCR specifically designed to pick up the exogenous but not the endogenous Eia mRNA. performed on wild-type Ela (A), Al variant (B) and A4 variant (C) 2.6 In vitro import of hurnan precursor PDH-Ela viii CHAPTER 3 Western blot analysis of plasmid expression of the wild-type and variant PDH-E1 a RT-PCRspecifically designed to generate a signal from the exogenous but not the endogenous Ela mRNA, performed on wild-type Ela(WT), the xo ce11 line, as well as H92D and H92N E ,avariants In vitro import of hurnan precursor PDH-Ela CHAPTER 4 Western blot analysis of plasrnid expression of the wiid-type and variant protein X peptides CHAPTER 5 Sequencing of plasmid clones containing PCR products amplified frorn the patient's cDNA Tissue distribution of mutated vs wild-type alleles of PDH-E la Western blot analysis of plasmid expression of the mutant R14 1 L and wild-type PDH-Elain the no ce11 line CHAPTER 6 Western blot analysis of the endogenous expression of the pynivate dehydrogenase complex (A). selected ho10 bc 1 complex proteins (B). and the bcl complex iron-sulphur protein ISP (C) 134 Ideograrn of human chromosome 2 showing the results of exclusion and deletion mapping for the rnitochondrial mulitenzyme de ficiency phenotype 135 CHAPTER 7 Functional motifs of PDH-Ela 154 LIST OF TABLES CHAPTER 2 Total PDH complex activities recorded and averaged for PDH-Ela variants transfected into the no ce11 CHAPTER 3 Mean total PDH complex activities given 2 SEM for controls.