Lee, Weissy M.

Mammalian acetyl-CoA carboxylase (ACC) isoforms, ACC-1 and ACC-2, catalyze the formation of malonyl-CoA, a substrate for fatty acid synthesis and fatty acyl chain elongation, and a potent inhibitor of beta-oxidation. Based on the phenotype of knockout mice, ACC contributes significantly to overall body energy metabolism and is a potential drug target for the treatment of obesity and type II diabetes. Highly-purified ACC-1 undergoes a dramatic increase in mass following treatment with allosteric activators such as citrate, apparently due to linear polymerization of ACC dimers. The current studies were undertaken to explore (a) the extent to which ACC-2 undergoes polymerization and (b) to investigate whether other cellular proteins associate with and perhaps regulate the polymerization process. Size exclusion chromatography and sucrose gradient sedimentation studies show that ACC-2 undergoes very modest changes in molecular size upon allosteric activation, although the extent of polymerization of ACC-2 is enhanced in the presence of ACC-1. Isolation of the largest molecular forms of ACC, followed by mass spectrometry analysis, reveals that ACC "polymers" are multi-protein complexes. The proteins most reliably detected in ACC polymer fractions include tubulin, actin, fatty acid synthase, and heat shock proteins. The significance of the additional components of ACC complexes is evaluated from effects on the kinetics of the ACC reaction and of the polymerization process, as well as by cellular colocalization using fluorescence microscopy and co-immunoprecipitation studies. [ABSTRACT FROM AUTHOR]

Mammalian isoforms of acetyl-CoA carboxylase (ACC-1 and ACC-2) play important roles in synthesis, elongation, and oxidation of long-chain fatty acids, and the possible significance of ACC in the development of obesity has led to interest in the development of inhibitors. Here, we demonstrate that pyridoxal phosphate (PLP) is a linear and reversible inhibitor of ACC-1 and ACC-2. ACC from rat liver and white adipose tissue (largely ACC-1) exhibited an IC50 of ∼200 μM, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 μM. ACC from rat liver was equally sensitive to PLP following extensive purification by avidin affinity chromatography. When added before citrate, PLP inhibited ACC with a Ki of ∼100 μM, reducing maximal activity >90% and increasing the Ka for citrate ∼5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by ∼4-fold. Inhibition of ACC was reversed by removal of PLP, either by washing or by reaction with hydroxylamine or amino-oxyacetate. ACC was irreversibly inhibited and radiolabeled, to a stoichiometry of ∼0.4 mol[H]/mol subunit, in the presence of PLP plus [³H]borohydride. Studies with structurally related compounds demonstrated that the reactive aldehyde and negatively charged substituents of PLP contribute importantly to ACC inhibition. The studies reported here suggest a rationale to develop ACC inhibitors that are not structurally related to the substrates or products of the reaction and an approach to probe the citrate-binding site of the enzyme. [ABSTRACT FROM AUTHOR]

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.