Crystal Structure and Substrate Specificity of Human Thioesterase 2: INSIGHTS INTO THE MOLECULAR BASIS FOR THE MODULATION OF FATTY ACID SYNTHASE.

The type I fatty acid synthase (FASN) is responsible for the de novo synthesis of palmitate. Chain length selection and release is performed by the C-terminal thioesterase domain (TE1). FASN expression is up-regulated in cancer, and its activity levels are controlled by gene dosage and transcriptional and post-translational mechanisms. In addition, the chain length of fatty acids produced by FASN is controlled by a type II thioesterase called TE2 (E.C. 3.1.2.14). TE2 has been implicated in breast cancer and generates a broad lipid distribution within milk. The molecular basis for the ability of the TE2 to compete with TE1 for the acyl chain attached to the acyl carrier protein (ACP) domain of FASN is unknown. Herein, we show that human TE1 efficiently hydrolyzes acyl-CoA substrate mimetics. In contrast, TE2 prefers an engineered human acyl-ACP substrate and readily releases short chain fatty acids from full-length FASN during turnover. The 2.8 Å crystal structure of TE2 reveals a novel capping domain insert within the α/ß hydrolase core. This domain is reminiscent of capping domains of type II thioesterases involved in polyketide synthesis. The structure also reveals that the capping domain had collapsed onto the active site containing the Ser-101-His-237-Asp-212 catalytic triad. This observation suggests that the capping domain opens to enable the ACP domain to dock and to place the acyl chain and 4'-phosphopantetheinyl-linker arm correctly for catalysis. Thus, the ability of TE2 to prematurely release fatty acids from FASN parallels the role of editing thioesterases involved in polyketide and non-ribosomal peptide synthase synthases.

Lipid-Free Apolipoprotein A-I Reduces Progression of Atherosclerosis by Mobilizing Microdomain Cholesterol and Attenuating the Number of CD131 Expressing Cells: Monitoring Cholesterol Homeostasis Using the Cellular Ester to Total Cholesterol Ratio.

BACKGROUND: Atherosclerosis is a chronic inflammatory disorder whose development is inversely correlated with high-density lipoprotein concentration. Current therapies involve pharmaceuticals that significantly elevate plasma high-density lipoprotein cholesterol concentrations. Our studies were conducted to investigate the effects of low-dose lipid-free apolipoprotein A-I (apoA-I) on chronic inflammation. The aims of these studies were to determine how subcutaneously injected lipid-free apoA-I reduces accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without sustained elevations in plasma high-density lipoprotein cholesterol concentrations. METHODS AND RESULTS: Ldlr-/- and Ldlr-/- apoA-I-/- mice were fed a Western diet for a total of 12 weeks. After 6 weeks, a subset of mice from each group received subcutaneous injections of 200 µg of lipid-free human apoA-I 3 times a week, while the other subset received 200 µg of albumin, as a control. Mice treated with lipid-free apoA-I showed a decrease in cholesterol deposition and immune cell retention in the aortic root compared with albumin-treated mice, regardless of genotype. This reduction in atherosclerosis appeared to be directly related to a decrease in the number of CD131 expressing cells and the esterified cholesterol to total cholesterol content in several immune cell compartments. In addition, apoA-I treatment altered microdomain cholesterol composition that shifted CD131, the common ß subunit of the interleukin 3 receptor, from lipid raft to nonraft fractions of the plasma membrane. CONCLUSIONS: ApoA-I treatment reduced lipid and immune cell accumulation within the aortic root by systemically reducing microdomain cholesterol content in immune cells. These data suggest that lipid-free apoA-I mediates beneficial effects through attenuation of immune cell lipid raft cholesterol content, which affects numerous types of signal transduction pathways that rely on microdomain integrity for assembly and activation.