Characterising the role of a putative mammalian aspartate dehydrogenase in hepatic lipogenesis and secretion of very low density lipoproteins

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. 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.