Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K d values and show them to range from ;10 ÿ6 M (for 4PU30) to 10 ÿ4 M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 Å resolution. Structurally, Vr CSBP is similar to plant pathogenesisrelated class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel b-sheet wrapped around a C-terminal a-helix, with two short a-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.

Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether dropletassociated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, ?80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with 14 C-labeled OA or palmitic acid resulted in [

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3-to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C.These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells. The appearance of cytosolic lipid droplets is observed in many cell types in physiological and pathological conditions. Adipocytes, the major lipid-storing cells in mammals, are full of lipid droplets. Foam cells in atherosclerotic lesions and parenchymal cells in fatty liver are lipid dropletcontaining cells associated with disease states. These cells accumulate massive amounts of cholesteryl ester and/or triacylglycerol (TG) as lipid droplets in their cytoplasmic space. Recently, we and others have shown that there is a distinct set of proteins specifically localized in lipid droplets (1-5), suggesting that the lipid droplet is an organized intracellular structure.Adipose differentiation-related protein (ADRP), also called adipophilin, is known to be a lipid droplet-associating protein. ADRP was originally found as a major protein induced in the early stages of adipocyte differentiation (6), although it is now known that ADRP is expressed in a variety of tissues and cells (7-13) in addition to macrophage-derived foam cells in atherosclerotic lesions (14). ADRP is the most abundant protein in lipid droplets in hepatic cells (3) and macrophages (Y. Fujimoto, et al., unpublished data).Expression of ADRP in macrophages during foam cell formation has been demonstrated by several studies. The gene-chip technique was used to show that ADRP is one of the most strongly induced genes in macrophages after incubation with oxidized LDL (OxLDL) (15). Addition of VLDL stimulated foam cell formation and ADRP mRNA expression in cultured macrophages (16,17). Expression of ADRP mRNA in human atherosclerotic lesions has been demonstrated by in situ hybridization experiments (14). Overexpression of the ADRP gene in cultured cells resulted in increased fatty a...

Various organisms store energy in a form of neutral lipids such as triacylglycerol (TG) and cholesteryl ester (CE). In mammals, these neutral lipids are intracellularly stored as typically seen in adipocytes, hepatocytes, steroidogenic cells and macrophage-derived foam cells. Storage of lipids has merits allows for energy preservation and synthesis of lipoproteins and steroid hormones. However, excess accumulation often leads to disorders such as obesity, fatty liver and atherosclerosis.1) It is therefore important to understand how neutral lipids are stored and mobilized intracellularly, as these mechanisms largely remain unclear. In animal cells, neutral lipids are deposited in intracellular compartments called lipid droplets (LDs, also called lipid storage droplets, lipid bodies or lipid particles). TG and CE are packed in LDs coated with phospholipid monolayer. 2,3)The liver is the center of lipid metabolism in the body. It recovers and synthesizes lipid molecules and reconstitutes them as lipoproteins such as very low-density lipoprotein (VLDL), which are secreted into the circulation and distributed throughout the body. Thus, hepatic cells are important stores of neutral lipids and act as a physiological buffer. Increased accumulation of neutral lipids often causes fatty liver disease accompanied by LD formation in the hepatocytes. Recently, attention has been focused on hepatic steatosis as a part of the metabolic syndrome since this is intimately related to other signs and symptoms of the syndrome including obesity, hypertension, insulin resistance, hyperglycemia and hyperlipidemia.4) Hepatic steatosis is also induced by factors other than nutrients, including drugs and toxic reagents. 5)In this study, we tested LD-induction activity of fatty acids and glucose, which can cause hepatic steatosis, using a human hepatocyte cell line; this suggested that long-chain free fatty acids (FFA) most effectively induce LDs. We also tested various compounds as to whether they could suppress LD formation induced by oleic acid (OA), a long chain FFA. The LD formation was effectively blocked by an inhibitor of long chain acyl-CoA synthetase (ACSL). These results suggest that long chain FFAs and ACSLs are significant factors in LD formation in hepatocytes. MATERIALS AND METHODSMaterials HuH7, a human hepatocyte cell line, was obtained from Health Science Research Resources Bank (cell no. JCRB0403; Osaka Japan). Fatty acids (as sodium salt) and 5-pregnen-3b-ol-20-one (pregnenolone) were purchased from Sigma (U.S.A.). Caffeine, capsaicin, carbon tetrachloride and dl-isoproterenol hydrochloride were purchased from Wako (Japan). Triacsin C was purchased from Kyowa Medex (Japan). 4,4,10b-Trimethyl-trans-decal-3b-ol (AMO 1618) was purchased from Calbiochem (U.S.A.). Anti-ADRP monoclonal antibody was purchased from PROGEN (Germany). Anti-17bHSD11 antiserum was prepared as described previously.6) Peroxidase-conjugated goat anti-IgG antibodies were purchased from Biosource International (Camarillo, California). ECL Western Blotti...