Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ϳ50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation. Acyl coenzyme A:1,2-diacylglycerol acyltransferase (DGAT)2 is a membrane-bound enzyme that catalyzes the biosynthesis of triacylglycerols (TGs) (1). TGs are a class of neutral lipid that represents the major form of stored energy in eukaryotic organisms (2). However, excessive accumulation of TG in tissues can lead to obesity and insulin resistance, whereas increased TG in the blood is a risk factor for atherosclerosis (3)(4)(5).DGAT catalyzes the formation of an ester bond between a long chain fatty acid (fatty acyl coenzyme A (CoA)) and the free hydroxyl group of diacylglycerol (DG), generating TG. Two DGAT genes have now been identified, Dgat1 and Dgat2, which share no sequence homology. DGAT1 belongs to a large family of membrane-bound O-acyltransferases (MBOAT) that includes acyl-CoA:cholesterol acyltransferase-1 and -2 (ACAT-1 and -2), which catalyze cholesterol ester biosynthesis (6 -10). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family including monoacylglycerol acylCoA acyltransferases 1-3 and wax synthase (9, 11-16). Biochemical analyses have indicated that DGAT1 and DGAT2 have distinct roles in TG metabolism. When overexpressed in cells, DGAT2 yielded a much larger increase in intracellular triacylglycerol than DGAT1 (17). In in vitro assays, DGAT1, but not DGAT2, was capable of using a broad array of acyl acceptors to synthesize diacylglycerol, retinyl, and wax esters in addition to triacylglycerol (18).In vivo experiments in mice have also provided strong evidence that DGAT1 and DGAT2 do not serve redundant roles in lipid metabolism. Dgat1-deficient (Dgat1 Ϫ/Ϫ ) mice were viable with modest reductions in TG con...

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acylCoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER. Triacylglycerols (TG),2 the major form of stored energy in eukaryotic organisms, are synthesized via the multistep glycerol 3-phosphate or Kennedy pathway (1). In this pathway, three fatty acyl-coenzyme A (CoA) molecules (activated forms of fatty acids) are linked via ester bonds to a glycerol backbone. The microsomal enzyme, acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final reaction of the pathway by forming an ester bond between a long chain fatty acid and the free hydroxyl group of diacylglycerol (DG) generating TG.Two different DGAT enzymes, DGAT1 and DGAT2, are responsible for the acyl-CoA-dependent synthesis of TG in mammalian tissues (2-4). The genes encoding these two enzymes have no sequence homology and they each belong to distinct gene families. DGAT1 belongs to a large family of membrane-bound O-acyltransferases that includes acyl-CoA: cholesterol acyltransferase (ACAT)-1 and -2, which catalyze cholesterol ester biosynthesis (5-9). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family that includes monoacylglycerol acyl-CoA acyltransferases 1-3 and wax synthase (3, 8, 10 -14). In mice and humans, DGAT1 and DGAT2 are expressed in most tissues, with the highest levels of expression found in tissues that are associated with TG metabolism (adipose tissue, liver, small intestine, and mammary gland) (3, 5).Studies in mice and in cells in culture have provided compelling evidence that DGAT2, more so than DGAT1, is responsible for the majority of TG synthesis and that these two enzymes have separa...

Anti-mitotic agents such as paclitaxel and docetaxel are widely used for the treatment of breast, ovarian and lung cancers. Although paclitaxel induces apoptosis, this drug also modulates autophagy. How autophagy affects paclitaxel activity, is unclear. We discovered that paclitaxel inhibited autophagy through two distinct mechanisms dependent on cell cycle stage. In mitotic cells, paclitaxel blocked activation of the class III phosphatidyl inositol 3 kinase, Vps34, a critical initiator of autophagosome formation. In non-mitotic paclitaxel-treated cells, autophagosomes were generated but their movement and maturation was inhibited. Chemically or genetically blocking autophagosome formation diminished paclitaxel-induced cell death suggesting that autophagosome accumulation sensitized cells to paclitaxel toxicity. In line with these observations, we identified that primary breast tumors that expressed diminished levels of autophagy-initiating genes were resistant to taxane therapy, identifying possible mechanisms and prognostic markers of clinical chemotherapeutic resistance.

Viral respiratory diseases remain problematic in swine. Among viruses, porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV), alone or in combination, are the two main known contributors to lung infectious diseases. Previous studies demonstrated that experimental dual infections of pigs with PRRSV followed by SIV can cause more severe disease than the single viral infections. However, our understanding of the impact of one virus on the other at the molecular level is still extremely limited. Thus, the aim of the current study was to determine the influence of dual infections, compared to single infections, in porcine alveolar macrophages (PAMs) and precision cut lung slices (PCLS). PAMs were isolated and PCLS were acquired from the lungs of healthy 8-week-old pigs. Then, PRRSV (ATCC VR-2385) and a local SIV strain of H1N1 subtype (A/Sw/Saskatchewan/18789/02) were applied simultaneously or with 3h apart on PAMs and PCLS for a total of 18 h. Immuno-staining for both viruses and beta-tubulin, real-time quantitative PCR and ELISA assays targeting various genes (pathogen recognition receptors, interferons (IFN) type I, cytokines, and IFN-inducible genes) and proteins were performed to analyze the cell and the tissue responses. Interference caused by the first virus on replication of the second virus was observed, though limited. On the host side, a synergistic effect between PRRSV and SIV co-infections was observed for some transcripts such as TLR3, RIG-I, and IFNβ in PCLS. The PRRSV infection 3h prior to SIV infection reduced the response to SIV while the SIV infection prior to PRRSV infection had limited impact on the second infection. This study is the first to show an impact of PRRSV/SIV co-infection and superinfections in the cellular and tissue immune response at the molecular level. It opens the door to further research in this exciting and intriguing field.

Background: DGAT2 catalyzes the final and committed step of triacylglycerol (TG) biosynthesis. Results: Co-immunoprecipitation experiments and in situ proximity ligation assays showed that DGAT2 and MGAT2 interact. Conclusion: DGAT2 can utilize diacylglycerol generated by MGAT2 for TG synthesis. Significance: These findings provide insight into the spatial arrangement of enzymes involved in triacylglycerol biosynthesis.