Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.     

Gonadal steroids and anterior lobe dynorphin in the male rat

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.     

Cell cycle changes in water properties in sea urchin eggs

This study concerned changes in the motional properties of cellular water during the first cell cycle of fertilized sea urchin eggs (Lytechinus variegatus). There was a significant decrease in proton NMR T1 relaxation time and in cytoplasmic ice crystal growth during mitosis and a significant increase in T1 time and cytoplasmic ice crystal size during cleavage. This was not caused by egg water content changes as reflected by egg volume measurements. Removal of both the fertilization membrane and the hyaline layer shortly after fertilization did not alter the pattern of T1 time changes at mitosis and cleavage as compared to whole eggs; thus, the pattern of T1 time changes was attributed to intracellular events. Treatment of fertilized eggs with cytochalasin B, an inhibitor of actin polymerization, did not block the fall in T1 time at mitosis, but did block cytokinesis and the increase in T1 time, which normally occurred at cleavage. A significant pattern of actin disassembly and reassembly at mitosis and cytokinesis was found by studies on the total amount of monomeric actin (G actin) using the DNase I assay. This led to the hypothesis that the observed changes in T1 time and ice crystal size during the first cell cycle were due to the depolymerization and polymerization of cytoplasmic actin. To test this, the effect of the in vitro polymerization of purified actin on the T1 time and on ice crystal growth was examined. It was concluded that changes in the T1 time and ice crystal growth upon polymerization of actin in vitro resembled the changes seen in vivo. These results suggest that changes in the motional properties of cytoplasmic water during the first cell cycle are due, at least in part, to the state of polymerization of cytoplasmic actin.     

Mechanisms of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants

The highly selective Type II glucocorticoid ligand RU28362 showed a clear biphasic effect on alpha-lactalbumin (alpha-LA) production in rat mammary gland explants, with a peak at 1 nM and a return to basal levels at 30-300 nM; dexamethasone showed a similar profile. Corticosterone, which has a higher affinity for Type I than Type II receptors, produced a variable response. In six out of eleven studies this was biphasic, with a maximum at 300 nM; in five no increase above baseline was seen. Classical Type I receptor ligands--aldosterone and deoxycorticosterone--showed responses parallel to their Type II agonist activity. We interpret these data as follows occupancy of Type I receptors does not increase alpha-LA production the response to selective Type II receptor ligands is truly biphasic and one explanation of this pattern may be the existence of both "turn-on" and "turn-off" acceptor sites in the nucleus.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.