Acat1/Soat1 knockout extends the mutant Npc1 mouse lifespan and ameliorates functional deficiencies in multiple organelles of mutant cells.

Multiple membrane organelles require cholesterol for proper function within cells. The Niemann-Pick type C (NPC) proteins export cholesterol from endosomes to other membrane compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), trans-Golgi network (TGN), and mitochondria, to meet their cholesterol requirements. Defects in NPC cause malfunctions in multiple membrane organelles and lead to an incurable neurological disorder. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), a resident enzyme in the ER, converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs in an NPC-independent manner. Here we report the interesting finding that in a mutant Npc1 mouse (Npc1nmf), Acat1 gene (Soat1) knockout delayed the onset of weight loss, motor impairment, and Purkinje neuron death. It also improved hepatosplenic pathology and prolonged lifespan by 34%. In mutant NPC1 fibroblasts, ACAT1 blockade (A1B) increased cholesterol content associated with TGN-rich membranes and mitochondria, while decreased cholesterol content associated with late endosomes. A1B also restored proper localization of syntaxin 6 and golgin 97 (key proteins in membrane trafficking at TGN) and improved the levels of cathepsin D (a key protease in lysosome and requires Golgi/endosome transport for maturation) and ABCA1 (a key protein controlling cholesterol release at PM). This work supports the hypothesis that diverting cholesterol from storage can benefit multiple diseases that involve cholesterol deficiencies in cell membranes.

Myeloid Acat1/Soat1 KO attenuates pro-inflammatory responses in macrophages and protects against atherosclerosis in a model of advanced lesions.

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1-M/-M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE-/-) mouse model for early lesions, Acat1-M/-M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1-M/-M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1-M/-M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1-M/-M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE-/- mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.

Myeloid-specific Acat1 ablation attenuates inflammatory responses in macrophages, improves insulin sensitivity, and suppresses diet-induced obesity.

Macrophages are phagocytes that play important roles in health and diseases. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) converts cellular cholesterol to cholesteryl esters and is expressed in many cell types. Unlike global Acat1 knockout (KO), myeloid-specific Acat1 KO ( Acat1-) does not cause overt abnormalities in mice. Here, we performed analyses in age- and sex-matched Acat1-M/-M and wild-type mice on chow or Western diet and discovered that Acat1-M/-M mice exhibit resistance to Western diet-induced obesity. On both chow and Western diets, Acat1-M/-M mice display decreased adipocyte size and increased insulin sensitivity. When fed with Western diet, Acat1-M/-M mice contain fewer infiltrating macrophages in white adipose tissue (WAT), with significantly diminished inflammatory phenotype. Without Acat1, the Ly6Chi monocytes express reduced levels of integrin-ß1, which plays a key role in the interaction between monocytes and the inflamed endothelium. Adoptive transfer experiment showed that the appearance of leukocytes from Acat1-M/-M mice to the inflamed WAT of wild-type mice is significantly diminished. Under Western diet, Acat1-M/-M causes suppression of multiple proinflammatory genes in WAT. Cell culture experiments show that in RAW 264.7 macrophages, inhibiting ACAT1 with a small-molecule ACAT1-specific inhibitor reduces inflammatory responses to lipopolysaccharide. We conclude that under Western diet, blocking ACAT1 in macrophages attenuates inflammation in WAT. Other results show that Acat1-M/-M does not compromise antiviral immune response. Our work reveals that blocking ACAT1 suppresses diet-induced obesity in part by slowing down monocyte infiltration to WAT as well as by reducing the inflammatory responses of adipose tissue macrophages.

Myeloid Acyl-CoA:Cholesterol Acyltransferase 1 Deficiency Reduces Lesion Macrophage Content and Suppresses Atherosclerosis Progression.

Acyl-CoA:cholesterol acyltransferase 1 (Acat1) converts cellular cholesterol to cholesteryl esters and is considered a drug target for treating atherosclerosis. However, in mouse models for atherosclerosis, global Acat1 knockout (Acat1(-/-)) did not prevent lesion development. Acat1(-/-) increased apoptosis within lesions and led to several additional undesirable phenotypes, including hair loss, dry eye, leukocytosis, xanthomatosis, and a reduced life span. To determine the roles of Acat1 in monocytes/macrophages in atherosclerosis, we produced a myeloid-specific Acat1 knockout (Acat1(-M/-M)) mouse and showed that, in the Apoe knockout (Apoe(-/-)) mouse model for atherosclerosis, Acat1(-M/-M) decreased the plaque area and reduced lesion size without causing leukocytosis, dry eye, hair loss, or a reduced life span. Acat1(-M/-M) enhanced xanthomatosis in apoe(-/-) mice, a skin disease that is not associated with diet-induced atherosclerosis in humans. Analyses of atherosclerotic lesions showed that Acat1(-M/-M) reduced macrophage numbers and diminished the cholesterol and cholesteryl ester load without causing detectable apoptotic cell death. Leukocyte migration analysis in vivo showed that Acat1(-M/-M) caused much fewer leukocytes to appear at the activated endothelium. Studies in inflammatory (Ly6C(hi)-positive) monocytes and in cultured macrophages showed that inhibiting ACAT1 by gene knockout or by pharmacological inhibition caused a significant decrease in integrin ß 1 (CD29) expression in activated monocytes/macrophages. The sparse presence of lesion macrophages without Acat1 can therefore, in part, be attributed to decreased interaction between inflammatory monocytes/macrophages lacking Acat1 and the activated endothelium. We conclude that targeting ACAT1 in a myeloid cell lineage suppresses atherosclerosis progression while avoiding many of the undesirable side effects caused by global Acat1 inhibition.

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids being trafficking into PC and PI. Collectively, these data support the conclusion that FATP2 has a dual function in the pathways linking the transport and activation of exogenous fatty acids. We discuss the differential roles of FATP2 and its role in both fatty acid transport and fatty acid activation in the context of lipid homeostasis.

Human fatty acid transport protein 2a/very long chain acyl-CoA synthetase 1 (FATP2a/Acsvl1) has a preference in mediating the channeling of exogenous n-3 fatty acids into phosphatidylinositol.

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M(r) 70,000) and FATP2b (M(r) 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14-C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.

Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae.

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% eta-dodecyl-beta-maltoside following expression at 15 degrees C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a Vmax of 158.2 nmol/min/mg protein and a Km of 71.1 microM oleate. When the concentration of oleate was held constant at 50 microM, the Km for CoA and ATP were 18.3 microM and 51.6 microM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.

Redox-sensitivity and site-specificity of S- and N- denitrosation in proteins.

BACKGROUND: S-nitrosation--the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins--is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated. METHODOLOGY/PRINCIPAL FINDINGS: The denitrosation of N-acetyl-nitroso Trp (NANT) by glutathione (GSH) required molecular oxygen and was inhibited by superoxide dismutase (SOD). Transnitrosation to form S-nitrosoglutathione (GSNO) was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA). Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO. CONCLUSIONS: We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.