Non-covalent control of mammalian acetyl-CoA carboxylase isoforms

Acetyl-CoA carboxylase (ACC) plays several crucial roles in lipid metabolism and has been identified as a potential drug target for the treatment of obesity and cancer. Many aspects of ACC structure, function and control remain unclear and my general goal was to provide new insights into two unresolved areas. The first part of this thesis describes work to characterize the key allosteric site that is responsible for citrate-induced ACC activation. The second area addressed is the hypothesis that protein-protein interactions provide critical aspects of ACC control. The effects of citrate on ACC were probed by studying the effects of pyridoxal phosphate (PLP), modeled on earlier studies of phosphofructokinase. A comprehensive assessment of the inhibition kinetics of ACC by PLP provides support for the effects of PLP being non-competitive with respect to substrates and therefore perhaps mediated by binding to a non-catalytic site. Further studies established methods for covalent attachment of PLP via Schiff base reduction with sodium borohydride and set the stage for more precise localization of PLP binding. To define proteins that interact specifically with ACC, techniques were established to separate large polymeric forms of ACC from smaller dimeric forms of the enzyme by size exclusion chromatography. Analysis of the citrate-induced ACC polymers by tandem mass spectrometry led to the identification of a small number of proteins that consistently associated with ACC in a citrate-dependent manner, including fatty acid synthase (FASN) and tubulin. The interactions of ACC with FASN and tubulin were further explored by Western blotting, co-immunoprecipitation, ACC activity assays and immunofluorescence analysis of primary rat hepatocytes. The evidence from several complementary experimental approaches supports specific interactions between ACC and tubulin. Tubulin appears to have subtle effects on ACC catalytic activity and may be more critical for structural organization and cellular localization of ACC. Immunofluorescence microscopy of ACC within primary rat hepatocytes led to novel insights, notably that ACC is localized into discrete structures rather than being distributed through the cytosolic compartment. This work opens the way to further characterization of the relevant subcellular structures and the role this localization plays in the control of ACC isoforms.