Hepatocytes play a crucial role in regulating lipid metabolism by exporting cholesterol and triglyceride into plasma through secretion of very low density lipoproteins (VLDL). VLDL production is also required for release of hepatitis C virus (HCV) from infected hepatocytes. Here, we show that long chain acyl-CoA synthetase 3 (ACSL3) plays a crucial role in secretion of VLDL and HCV from hepatocytes. In cultured human hepatoma Huh7 cells, ACSL3 is specifically required for incorporation of fatty acids into phosphatidylcholine. In cells receiving small interfering RNA targeting ACSL3, secretion of apolipoprotein B, the major protein component of VLDL, was inhibited and the lipoprotein was rapidly degraded. This inhibition in secretion was completely eliminated when these cells were treated with phosphatidylcholine. Treatment of cells with small interfering RNA targeting ACSL3 also inhibited secretion of HCV from Huh7-derived cells. These results identify ACSL3 as a new enzymatic target to limit VLDL secretion and HCV infection.
"Among the different members of the ACSL family, member 3 and 6 have a strong preference for activation of long-chain fatty acids toward synthesis of the glycerophospholipid phosphatidylcholine , . ACLS3 and ACSL6 are membrane proteins associated with different organelles, such as the ER and Golgi apparatus , . "
[Show abstract][Hide abstract] ABSTRACT: Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen that multiplies within a parasitophorous vacuole called an inclusion. We report that the location of several host-cell proteins present in the cytosol, the nucleus, and membranes was altered during Ct development. The acyl-CoA synthetase enzyme ACSL3 and the soluble acyl-CoA binding protein ACBD6 were mobilized from organelle membranes and the nucleus, respectively, into the lumen of the inclusion. The nuclear protein ZNF23, a pro-apoptosis factor, was also translocated into the inclusion lumen. ZNF23, among other proteins, might be targeted by Ct to inhibit host cell apoptosis, thereby enabling bacterial survival. In contrast, the acyl-CoA:lysophosphatidylcholine acyltransferase LPCAT1, an endoplasmic reticulum membrane protein, was recruited to the inclusion membrane. The coordinated action of ACBD6, ACSL3 and LPCAT1 likely supports remodeling and scavenging of host lipids into bacterial-specific moieties essential to Ct growth. To our knowledge, these are the first identified host proteins known to be intercepted and translocated into the inclusion.
PLoS ONE 05/2012; 7(5):e36843. DOI:10.1371/journal.pone.0036843 · 3.23 Impact Factor
"The importance of the PC biosynthetic pathway in HCV replication has been shown previously; one recent study demonstrated a progressive accumulation of several PC species during HCV infection . In addition, Yao et al demonstrated that the enzyme, long chain acyl-CoA synthetase 3 (ACSL3), is specifically required for incorporation of fatty acids into phosphatidylcholine and treatment of cells with siRNA targeting ACSL3 inhibited the secretion of VLDL and HCV particles from hepatocytes . PC synthesis is required for the normal secretion of VLDL by hepatocytes  and it has previously been suggested that HCV uses the VLDL assembly and secretion pathway for maturation and secretion of viral particles , . "
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 5'-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes.
PLoS ONE 08/2011; 6(8):e23641. DOI:10.1371/journal.pone.0023641 · 3.23 Impact Factor
"The protein repertoire of CLDs included ACSL3 and ACSL4, which are responsible for the activation of fatty acids prior to their entry into metabolic pathways, such as de novo lipid synthesis, fatty acid catabolism or remodelling of membranes. In human Huh7 hepatocytes, ACSL activity has been involved in local synthesis of neutral lipids in lipid droplets (Fujimoto et al., 2007) and, more recently, ACSL3, which is the most abundant ACSL in Caco-2/TC7 enterocyte lipid droplet proteome, has been shown to be specifically required for fatty acid incorporation into PC, an essential reaction for VLDL (very-low-density lipoprotein) assembly, the hepatic form of TRL (Yao and Ye, 2008). Although major differences exist, it is interesting to note that the complex and highly differentiated function of TRL assembly and secretion is shared by hepatocytes and enterocytes. "
[Show abstract][Hide abstract] ABSTRACT: Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.
Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.
We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
Biology of the Cell 07/2011; 103(11):499-517. DOI:10.1042/BC20110024 · 3.51 Impact Factor
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