Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol. acid with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyrindine in dichloromethane. After reaction, 25-HC oleate was purified by preparative TLC on silica gel (10:1 hexane:ethyl acetate). Chemical structure was determined by the NMR analysis and MS. 25-HC [1-14C]oleate was synthesized from 25-HC and [1-14C]oleic acid. Lipoproteins acLDL and lipoprotein deficient serum (LPDS) were prepared as described previously (28). Animals Mice lacking ((for 45 min at 4C, microsomal pellet was resuspended and re-ultracentrifuged to enhance purity to give a supernatant fraction (cysotol) and a microsomal pellet (22, 23). TLC Lipid was extracted from the cytosolic (100 g of protein) and microsomal fraction (50 g of protein), and was separated by TLC with toluene-ethyl acetate (67:33) as the solvent. Visualization was done with 10% sulfuric acid. Measurements of oxysterols Concentrations of oxysterols in subcellular fractions were measured using LC-MS/MS as described (32). After the addition of deuterated internal standards and butylated hydroxytoluene, each fraction was either hydrolyzed with 1 N ethanolic KOH and derivatized into picolinyl esters, or directly converted into picolinyl esters. Northern blot analysis Northern blot analyses were performed as described (9). Analysis of Xbp-1 mRNA splicing Total RNA was reverse transcribed and amplified using a sense primer (5-AAACAGAGTAGCAGCGCAGACTGC-3) and an antisense primer (5-GGATCTCTAAAACTAGAGGCTTGGTG-3). This fragment was further digested by as described previously (33). Quantitative real-time PCR Two micrograms of total RNA were reverse-transcribed using the ThermoScript RT-PCR system (Invitrogen). Quantitative real-time PCR was performed using SYBR Green dye (Applied Biosystems, Foster City, CA) in an ABI Prism 7900 PCR instrument (Applied Biosystems). The relative abundance of each transcript was calculated from a standard curve of cycle thresholds for serial dilutions of a cDNA sample and normalized to or and 3-hydroxy-3-methylglutaryl-CoA synthase 1 ((sense 5-AGCCTGCAGTTTGAGCTTA-3, antisense 5-AGA-GT-CGGTATTTCTGGAGACG-3), (sense 5-GGAAGTTGG-GTGCCACTTCG-3, antisense 5-GGTGCTCTCAGATCTTTGG-3), (sense 5-GAAGACAGGGCGACCTG-GA-A-3, antisense 5-T-T-GTGGCTCCCACAATGAAGC-3), and (sense 5-CGATGCCCT-GAGGCTCTTT-3, antisense 5-TG-GATGCCACAGGATTCCA-3). Western blot analyses TGEMs were homogenized in buffer A [50 mM Tris-HCl, 250 mM sucrose, 1 mM EDTA, 2 g/ml leupeptin (pH 7.0)]. Ten micrograms of proteins of whole lysates were separated by SDS-PAGE around the NuPAGE 10% Bis-Tris gel and transferred to a nitrocellulose membrane. For detection of the proteins, the membranes were incubated with each anti-murine Akt (Abcam) or anti-murine GAPDH at a dilution of 1 1:1,000 in Hikari A solution (Nacalai Tesque). Specifically bound immunoglobulins were detected in a second reaction with a horseradish peroxidase-labeled IgG conjugate and visualized by ECL detection (GE Healthcare) with Image Quant LAS 4000 Mini (GE Healthcare). Statistics Statistical differences between groups were analyzed by one-way ANOVA and the post hoc Tukey-Kramer test or two-tailed Students deficiency increases the susceptibility of macrophages to apoptosis While attempting to examine the intracellular structures of the 0.05; ** 0.01. Open in a separate window Fig. 2. Inhibition of ACAT1 suppresses the augmentation of 25-HC-induced apoptosis in was measured by RT-PCR. Data are expressed as the mean SEM. ** 0.01; NS, a nonsignificant difference. The augmentation of the 25-HC-induced apoptosis, which was clearly observed in than the nonelicited WT cells (supplementary Fig. IA), there was no significant difference in the number of apoptotic cells between the two types of cells after treatment with 25-HC (supplementary Fig. IB). These results indicate that nonelicited macrophages are more susceptible to 25-HC-induced ER stress and apoptosis than elicited TGEMs. The susceptibility may be conferred by both the lower expression of and higher expression of (supplementary Fig. IC, D). ACAT inhibitors suppress Nceh1-dependent macrophage apoptosis Membrane-bound enzyme ACAT, which is responsible for the intracellular esterification of cholesterol, is known to be strongly activated by oxysterols (35). Inasmuch as we have reported that Nceh1 catalyzes the intracellular hydrolysis of CE (2), we speculated that Nceh1 also catalyzes the intracellular hydrolysis of esterified oxysterols and that cycles of esterification-hydrolysis could play a pivotal Ac-LEHD-AFC role in the underlying molecular processes. Indeed, Nceh1 hydrolyzed 25-HC oleate in vitro (supplementary Fig. II). of Nceh1 for 25-HC oleate was comparable to that for cholesteryl oleate: 6.4 0.9 M versus 6.9 2.3 M. As expected, nonselective ACAT inhibitor, CS-505, significantly inhibited the Nceh1-dependent augmentation of enhanced 25-HC-induced apoptosis (Fig. 2A). There are two ACAT isozymes: ACAT1 and ACAT2. To determine which isozyme mediated the 25-HC-induced apoptosis, we further compared the effects of ACAT1-specific inhibitor, K-604, and ACAT2-specific inhibitor, PPPA. As expected, the augmentation of the 25-HC-induced apoptosis was spe-cifically inhibited by K-604, but not by PPPA, corroborating the fact that ACAT1 is the major isozyme of TGEMs (36). A similar phenomenon was also observed for 7-KC. Microsomal accumulation of 25-HC ester precedes activation of ER stress signaling in genotype. The induction of the expression was inhibited by K-604 and CS-505, but.E., Rusinol A. synthesized from 25-HC and [1-14C]oleic acid. Lipoproteins acLDL and lipoprotein deficient serum (LPDS) were prepared as described previously (28). Animals Mice lacking ((for 45 min at 4C, microsomal pellet was resuspended and re-ultracentrifuged to enhance purity to give a supernatant fraction (cysotol) and a microsomal pellet (22, 23). TLC Lipid was extracted from the cytosolic (100 g of protein) and microsomal fraction (50 g of protein), and was separated by TLC with toluene-ethyl acetate (67:33) as the solvent. Visualization was done with 10% sulfuric acid. Measurements of oxysterols Concentrations of oxysterols in subcellular fractions were measured using LC-MS/MS as described (32). After the addition of deuterated internal standards and butylated hydroxytoluene, each fraction was either hydrolyzed with 1 N ethanolic KOH and derivatized into picolinyl esters, or directly converted into picolinyl esters. Northern blot analysis Northern blot analyses were performed as described (9). Analysis of Xbp-1 mRNA splicing Total RNA was reverse transcribed and amplified using a sense primer (5-AAACAGAGTAGCAGCGCAGACTGC-3) and an antisense primer (5-GGATCTCTAAAACTAGAGGCTTGGTG-3). This fragment was further digested by as described previously (33). Quantitative real-time PCR Two micrograms of total RNA were reverse-transcribed using the ThermoScript RT-PCR system (Invitrogen). Quantitative real-time PCR was performed using SYBR Green dye (Applied Biosystems, Foster City, CA) in an ABI Prism 7900 PCR instrument (Applied Biosystems). The relative abundance of each transcript was calculated from a standard curve of cycle thresholds for serial dilutions of a cDNA sample and normalized to or and 3-hydroxy-3-methylglutaryl-CoA synthase 1 ((sense 5-AGCCTGCAGTTTGAGCTTA-3, antisense 5-AGA-GT-CGGTATTTCTGGAGACG-3), (sense 5-GGAAGTTGG-GTGCCACTTCG-3, antisense 5-GGTGCTCTCAGATCTTTGG-3), (sense 5-GAAGACAGGGCGACCTG-GA-A-3, antisense 5-T-T-GTGGCTCCCACAATGAAGC-3), and (sense 5-CGATGCCCT-GAGGCTCTTT-3, antisense 5-TG-GATGCCACAGGATTCCA-3). Western blot analyses TGEMs were homogenized in buffer A [50 mM Tris-HCl, 250 mM sucrose, 1 mM EDTA, 2 g/ml leupeptin (pH 7.0)]. Ten micrograms of proteins of whole lysates were separated by SDS-PAGE Ac-LEHD-AFC around the NuPAGE 10% Bis-Tris gel and transferred to a nitrocellulose membrane. For detection of the proteins, the membranes were incubated with each anti-murine Akt (Abcam) or anti-murine GAPDH at a dilution of 1 1:1,000 in Hikari A solution (Nacalai Tesque). Specifically bound immunoglobulins Mouse monoclonal antibody to HAUSP / USP7. Ubiquitinating enzymes (UBEs) catalyze protein ubiquitination, a reversible process counteredby deubiquitinating enzyme (DUB) action. Five DUB subfamilies are recognized, including theUSP, UCH, OTU, MJD and JAMM enzymes. Herpesvirus-associated ubiquitin-specific protease(HAUSP, USP7) is an important deubiquitinase belonging to USP subfamily. A key HAUSPfunction is to bind and deubiquitinate the p53 transcription factor and an associated regulatorprotein Mdm2, thereby stabilizing both proteins. In addition to regulating essential components ofthe p53 pathway, HAUSP also modifies other ubiquitinylated proteins such as members of theFoxO family of forkhead transcription factors and the mitotic stress checkpoint protein CHFR were detected in a second reaction with a horseradish peroxidase-labeled IgG conjugate and visualized by ECL detection (GE Healthcare) with Image Quant LAS 4000 Mini (GE Healthcare). Statistics Statistical differences between groups were analyzed by one-way ANOVA and the post hoc Tukey-Kramer test or two-tailed Students deficiency increases the susceptibility of macrophages to apoptosis While attempting to examine the intracellular structures of the 0.05; ** 0.01. Open in a separate window Fig. 2. Inhibition of ACAT1 suppresses the augmentation Ac-LEHD-AFC of 25-HC-induced apoptosis in was measured by RT-PCR. Data are expressed as the mean SEM. ** 0.01; NS, a nonsignificant difference. The augmentation of the 25-HC-induced apoptosis, which was Ac-LEHD-AFC clearly observed in than the nonelicited WT cells (supplementary Fig. IA), there was no significant difference in the number of apoptotic cells between the two types of cells after treatment with 25-HC (supplementary Fig. IB). These results indicate that nonelicited macrophages are more susceptible to 25-HC-induced ER stress and apoptosis than elicited TGEMs. The susceptibility may be conferred by both the lower expression of and higher expression of (supplementary Fig. IC, D). ACAT inhibitors suppress Ac-LEHD-AFC Nceh1-dependent macrophage apoptosis Membrane-bound enzyme ACAT, which is responsible for the intracellular esterification of cholesterol, is known to be strongly activated by oxysterols (35). Inasmuch as we have reported that Nceh1 catalyzes the intracellular hydrolysis of CE (2), we speculated.