Considerable evidence supports the involvement of acyl-CoA:cholesterol acyltransferase (ACAT) in the maintenance of intracellular cholesterol homeostasis. A number of recently developed ACAT inhibitors may have potential use as pharmacological agents to reduce the development of atherosclerosis. Recently, however, reports arose describing cytotoxic effects following administration of a specific ACAT inhibitor to experimental animals. In order to address the specific intracellular mechanisms involved with the cytotoxic effect, we examined the consequences of ACAT inhibition in cholesterol-enriched mouse peritoneal macrophages. Mouse peritoneal macrophages were cholesterol-enriched by incubation with acetylated low density lipoprotein and free cholesterol:phospholipid dispersions prior to the addition of an ACAT inhibitor, either Sandoz 58-035 or Pfizer CP-113,818. The adenine pool of the macrophages was radiolabeled prior to addition of the ACAT inhibitors, in order to monitor the release of radiolabeled adenine, a technique shown to be a sensitive method to monitor drug-induced toxicity. The ACAT inhibitors were added for up to 48 h and at concentrations up to 2 micrograms/ml. These conditions resulted in an approximately 2-fold increase in adenine release. The increase in cell toxicity paralleled an increase in the cellular free cholesterol content. Reducing the cellular free cholesterol content, by the addition of extracellular acceptors, decreased the cytotoxic effects of the ACAT inhibitors. Addition of an intracellular cholesterol transport inhibitor, either progesterone or U18666A, together with CP-113,818 blocked the toxic effect of CP-113,818. These results suggest that ACAT inhibition of cholesterol-enriched macrophages increases cell toxicity due to the buildup of cellular free cholesterol. Removal of free cholesterol by the addition of extracellular cholesterol acceptors or by blocking intracellular sterol transport relieves the ACAT inhibitor-induced toxicity.
A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.
Abstract-This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl--cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment.
Kinases represents one of the most important family of targets in high throughput drug screening. Tyrosine kinases and serine/threonine kinases are known to play key roles in signal transduction as well as in cell growth and differentiation. Intense screening campaigns are underway in all major pharmaceuticals and large biotech companies to find kinase inhibitors for the treatment of inflammatory diseases, immunological disorders and cancer. The present contribution describes models that were developed to produce kinase assays amenable to HTS using AlphaScreen. Because of the flexibility allowed by AlphaScreen, kinase assays can be developed using direct or indirect approaches. Tyrosine kinase assays are usually performed with a direct format involving generic anti-phosphotyrosine antibodies while serine/threonine kinase assays are performed with an indirect format where specific antibodies are captured using protein A conjugated Acceptor beads. Streptavidin-coated Donor beads are used to capture either generic (ex. poly GT) or specific biotinylated substrates. Herein, are presented different methods to perform screening for inhibitors acting on the soluble beta-insulin receptor tyrosine kinase (IRKD), and on p38, a member of the MAP kinase family.
The cytokines CLCF1 and CNTF are ligands for the CNTF receptor and the apolipoprotein E (ApoE) receptor sortilin. Both share structural similarities with the N-terminal domain of ApoE, known to bind CNTF. We therefore evaluated whether ApoE or ApoE-containing lipoproteins interact with CLCF1 and regulate its activity. We observed that CLCF1 forms complexes with the three major isoforms of ApoE in co-immunoprecipitation and proximity assays. FPLC analysis of mouse and human sera mixed with CLCF1 revealed that CLCF1 co-purifies with plasma lipoproteins. Studies with sera from ApoE−/− mice indicate that ApoE is not required for CLCF1-lipoprotein interactions. VLDL- and LDL-CLCF1 binding was confirmed using proximity and ligand blots assays. CLCF1-induced STAT3 phosphorylation was significantly reduced when the cytokine was complexed with VLDL. Physiological relevance of our findings was asserted in a mouse model of oxygen-induced retinopathy, where the beneficial anti-angiogenic properties of CLCF1 were abrogated when co-administrated with VLDL, indicating, that CLCF1 binds purified lipoproteins or lipoproteins in physiological fluids such as serum and behave as a “lipocytokine”. Albeit it is clear that lipoproteins modulate CLCF1 activity, it remains to be determined whether lipoprotein binding directly contributes to its neurotrophic function and its roles in metabolic regulation.
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