Characterising the Role of a Putative Mammalian Aspartate Dehydrogenase in Hepatic Lipogenesis and Secretion of Very Low Density Lipoproteins STEPHANIE ANN BONNEY Thesis submitted to the Imperial College of London For the degree of Doctor of Philosophy December 2009 MRC Genomic and Molecular Medicine Clinical Sciences Centre Imperial College School of Medicine Hammersmith Hospital, Du Cane Road London W12 ONN, UK. 1 i Acknowledgements I owe much gratitude to my Ph.D. supervisor, Professor Carol Shoulders for providing me the opportunity, support, advice, guidance and strength to undertake and complete a Ph.D. I would like to thank the many people who have significantly contributed to my studies and helped me along my way: Rocio Lale-Montes, Angela Whyte, Christopher Mann, Penny Ritchie, Abdul Hebbachi, Geoffrey Gibbons, Bethan Jones, Emma Jones and Max Salm. I am indebted to my many colleagues for providing a stimulating and fun environment in which to learn and grow. I am especially grateful to Helen Ringham, Claire Hutchison, Ruth Newton, Margaret Town, Paul Williams, Muddassar Mirza, Stuart Horswell and Lee Fryer. I wish to thank my family and friends for helping me get through the difficult times, and for all the emotional support, comradery, entertainment, and caring they provided. Especially to my mother Sylvia Bonney, who provided consistent support and encouragement through-out the many years of my Ph.D. To my brothers Craig, John and Sean, my sister in-laws Tracey, Francine and Suey and to all my nieces and nephews thank you for being there and supporting me through out the years. To all my friends, especially Denise Read, Victoria Westhead, Julia Barnett and Sophie Voline who have all been so supporting and caring through out the whole experience. Lastly, and most importantly, I wish to thank my husband, James Morton for giving the support to see the Ph.D. through to completion and for proof-reading all 251 pages of the thesis. I love you with all my heart. I dedicate this thesis to my father William Raymond Bonney, whose loving memory has guided my through the tough times and provided me with the strength to complete - you were my leading light. 2 ii Preface Publications relevant to this thesis are; Griffin,J.L., Bonney,S.A., Mann,C., Hebbachi,A.M., Gibbons,G.F., Nicholson,J.K., Shoulders,C.C., and Scott,J. (2004). An integrated reverse functional genomic and metabolic approach to understanding orotic acid-induced fatty liver. Physiol Genomics 17, 140-149. Hebbachi,A.M., Seelaender,M.C., Baker,B.W., and Gibbons,G.F. (1997). Decreased secretion of very-low-density lipoprotein triacylglycerol and apolipoprotein B is associated with decreased intracellular triacylglycerol lipolysis in hepatocytes derived from rats fed orotic acid or n-3 fatty acids. Biochem. J. 325 ( Pt 3), 711-719. Yang,Z., Savchenko,A., Yakunin,A., Zhang,R., Edwards,A., Arrowsmith,C., and Tong,L. (2003). Aspartate dehydrogenase, a novel enzyme identified from structural and functional studies of TM1643. J. Biol. Chem. 278, 8804-8808. 3 iii Abstract Hepatic lipogenesis and the secretion of very low-density lipoproteins are severely perturbed in orotic-acid fed rats. Proteomic analysis of this rodent fatty liver identified a protein enriched in the microsomal fraction. The protein contains a domain of unknown function 108 (DUF 108) and is found to compose a complete protein in archaebacteria and mammalian species, but a single domain in Caenorhabditis elegans protein Q19527. The cDNA for human protein A6ND91 was cloned and stable rat hepatoma cell lines, expressing recombinant A6ND91-FLAG tagged protein were produced. Significant increases in de-novo lipogenesis and secretion of triglyceride-enriched lipoproteins were observed. This thesis considers A6ND91 protein in terms of structure and proposed function. Previous work has indicated that the homologous protein Thermatoga maritime 1643 (TM1643) possesses NAD+ or NADP+-dependent dehydrogenase activity towards L-Aspartate, producing NADH or NADPH plus iminoaspartate, a substrate that rapidly degrades into oxaloacetate (OAA) and ammonia. In this thesis, analyses of primary and predicted secondary structure of A6ND91 and TM1643 revealed that residues binding NAD+/NADP+ and the proposed active catalytic residue, Histidine198, are conserved. Northern-blot analysis identified A6ND91 is highly expressed in human liver, moderately in kidney, and at low levels in the brain. Western-blot analysis identified that A6ND91 is predominately localised to the cytosol, with minimal amounts in the microsomal fraction. Proteomic analysis revealed that A6ND91 contains a phosphorylated residue, Serine168. The kinase potentially responsible for phosphorylation of the residue is Glycogen Synthase Kinase 3 (GSK3), a well- characterised enzyme involved in regulation of glycogen metabolism and acetyl-CoA production. Microarray analyses indicate that over-expression of A6ND91 perturbs activity of several metabolic pathways involved in carboxylic acid metabolism, cholesterol synthesis and complement activation. Closer inspection of the perturbed metabolic pathways revealed an overlap through the substrates OAA/pyruvate/acetyl-CoA. 4 Assays were performed to establish the NAD+/NADH and NADP+/NADPH redox state in cell lines expressing A6ND91. Results established that NAD+/NADH ratio was significantly reduced, suggesting that A6ND91 may operate as a dehydrogenase. Collectively, results suggest that L-Aspartate dehydrogenase activity of TM1643 is conserved in human A6ND91, playing a key role in hepatic lipid and energy metabolism through the production of OAA and its subsequent conversion to pyruvate/acetyl-CoA and cholesterol. 5 iv Contents Page No. i Acknowledgements 2 ii Preface 3 iii Abstract 4 iv Contents 6 v List of Figures 12 vi List of Tables 16 vii List of Appendices 18 viii Abbreviations 19 1.0 Chapter 1 Introduction 22 1.1 General Introduction 22 1.2 Metabolism of Amino Acids and Impact on Lipogenesis 22 1.3 Orotic Acid and Fatty Liver 32 1.4 Identification of Mammalian DUF 108 Protein 36 1.5 Aims 42 2.0 Chapter 2. Materials and Methods 43 2.1 General Molecular and Protein Materials and Methods 43 2.1.1 Human and Rat Tissue 43 2.1.2 Generation of Rabbit Polyclonal Human A6ND91-FLAG 43 Antibody 2.1.3 Cellular Homogenisation 44 2.1.4 Protein Solubilisation and Quantification 45 6 2.1.5 Shrimp Alkaline Phosphatase (SAP) Assay 45 2.1.6 SDS-PAGE 45 2.1.7 Two-Dimensional (2D) Iso-Electric Focusing (IEF) 46 2.1.8 Matrix-Assisted Laser Desorption Ionization (MALDI) MS 46 2.1.9 HPLC Electrospray Tandem MS 47 2.1.10 Western Blotting 47 2.2 Characterisation of Human A6ND91-FLAG 48 2.2.1 Generation of Human A6ND91-FLAG Probe 48 2.2.2 Probe Labelling and Northern Blot 50 2.2.3 In-vitro Expression of Human A6ND91-FLAG 51 2.2.4 NAD: NADH and NADP: NADPH Assays 52 2.2.5 ATP: ADP Assays 53 2.3 Cloning and Expression of EDM07494-FLAG 54 2.3.1 Construction of EDM07494-FLAG cDNA 54 2.3.2 Purification of Amplified EDM07494-FLAG cDNA 55 2.3.3 Restriction Enzyme Digestion of EDM07494-FLAG cDNA and 55 PcDNA4/TO Vector 2.3.4 Ligation of PcDNA4/TO vector EDM07494-FLAG cDNA 55 2.3.5 Transformation of TOP10 bacterial cells with Plasmid DNA 56 2.3.6 Growth of Transformed Bacterial Colonies 56 2.3.7 Screening Constructs for EDM07494-FLAG Insert 58 2.3.8 High Yield Growth and Purification of EDM07494-FLAG 58 Construct 7 2.3.9 Stable Cell line Rat 58 2.3.10 Isolation of Colonies 59 2.4 Microarray Methods 59 2.4.1 RNA Extraction 59 2.4.2 Quantitative and Qualitative Assessment of RNA 60 2.4.3 First-Strand cDNA synthesis 61 2.4.4 Second-Strand cDNA synthesis 61 2.4.5 Preparation of cRNA 62 2.4.6 Hybridisation and Staining Procedures 62 2.4.7 GeneChip Wash and Staining Procedure 63 2.4.8 Analysis of Gene Chip Data 63 2.4.9 Principle Component Analysis (PCA) 64 2.4.10 Identifying Differentially Expressed Genes 65 2.4.11 Annotation of Unknown Expressed Sequence Tags (ESTs) 65 2.4.12 Gene Ontology (GO) 65 2.4.13 Real Time RT-PCR 65 2.1.14 KEGG PATHWAY 66 3.0 Chapter 3. Characterisation of Human A6ND91 and Rat EDM07494 68 3.1 Introduction 68 3.2 Results 75 3.2.1 Protein Alignment of DUF 108 family 75 3.2.2 Expression of A6ND91 in Human Tissue 75 8 3.2.3 Characterization of Human A6ND91-FLAG Polyclonal 75 Antibody 3.2.4 In vitro expression of A6ND91-FLAG and Rat EDM07494- 79 FLAG 3.2.5 Post Translational Modification of Human A6ND91-FLAG and 83 Rat EDM07494-FLAG 3.2.6 A6ND91 is Predominantly Cytosolic 91 3.2.7 NAD: NADH and NADP: NADPH assay 91 3.2.8 ATP: ADP Ratio in A6ND91-FLAG expressing Cells 94 3.3 Discussion 98 4.0 Chapter 4. Microarray and RTPCR in Cells Expressing A6ND91 104 4.1 Introduction 104 4.2 Results 107 4.2.1 Expression of Human A6ND91-FLAG as identified by 107 Western Blot 4.2.2 Quality Control of total RNA, cDNA and cRNA 107 4.2.3 Hybridisation to Affymetrix GeneChip Rat Genome 230 2.0 112 Array 4.2.4 Quality of Hybridisation 112 4.2.5 Normalisation of data using CARMAWeb 119 4.2.6 Differentially expressed probe sets identified by Principle 119 Component Analysis (PCA) and Significance Analysis of Microarrays (SAM). 4.2.7 Human A6ND91-FLAG RNA expression 122 4.2.8 Functional annotation 122 9 4.2.9 GO Classification 122 4.2.10 Real Time RT-PCR 124 4.3 Discussion 129 5.0 Chapter 5. Perturbed Metabolic Pathways in Cells Expressing 134 A6ND91 5.1 Introduction 134 5.2 Results 136 5.2.1 Mapping Differentially Expressed Genes to Metabolic 136 Pathways 5.2.2 Cholesterol and Steroid Metabolism 136 5.2.3 Xenobiotic Metabolism 143 5.2.4 Carboxylic Acid Metabolism 146 5.2.4.1 Amino Acid Metabolism 157 5.2.4.2 Alanine and Aspartate 157 5.2.4.3.