Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists.

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.

Differential regulation of HIF-1 alpha prolyl-4-hydroxylase genes by hypoxia in human cardiovascular cells.

Three HIF-alpha prolyl-4-hydroxylases (PHDs) (named PHD1, PHD2, and PHD3) effect the proteasome-mediated degradation of HIF by catalyzing the hydroxylation of key proline residues in the HIF-1 alpha subunit under normoxic conditions. When oxygen tension is reduced, PHD-mediated hydroxylation cannot occur, HIF-1 alpha accumulates in the nucleus, resulting in HIF-mediated gene transcription. In the present study, the expression and regulation of PHD mRNA and HIF protein expression was examined in human tissues and primary cells of cardiovascular origin. Treatment of human cardiac myocytes, smooth muscle cells, and endothelial cells with hypoxia or CoCl(2), a hypoxia mimic, resulted in a significant time-dependent increase in PHD3, but not PHD1 or PHD2, mRNA levels, which correlated with an increase in HIF-1 alpha protein expression. Overexpression studies revealed that PHD3 levels influence HIF-1 alpha stability in both normoxic and hypoxic conditions, suggesting that PHD3 may participate in a feedback loop controlling HIF activity.

SDP1 is a peroxisome-proliferator-activated receptor gamma 2 co-activator that binds through its SCAN domain.

Peroxisome-proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, play an important role in the regulation of lipid metabolism and energy homoeostasis. In a yeast two-hybrid experiment using the zinc-finger transcription factor ZNF202 as bait, we previously identified the SCAN-domain-containing protein SDP1. SDP1 shares a high degree of amino acid sequence identity with PGC-2, a previously identified PPAR gamma 2 co-activator from the mouse. Here we show that SDP1 and PGC-2 interact with PPAR gamma 2 through their SCAN domains, even though PPAR gamma 2 does not contain a SCAN domain. Similar to PGC-2, SDP1 enhanced PPAR gamma 2-dependent gene transcription in transiently transfected cells but did not alter the affinity of PPAR gamma 2 for agonists. Although the SCAN domain was necessary for binding to PPAR gamma 2, it was not sufficient for co-activation in cells, suggesting that other features of SDP1 are responsible for transcriptional co-activation. The ability of SDP1 to interact with two different transcription factors that regulate genes involved in lipid metabolism, ZNF202 and PPAR gamma 2, suggests that SDP1 may be an important co-regulator of such genes.

Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a.

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture. Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-microl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.

An mRNA splice variant of the AFX gene with altered transcriptional activity.

Several studies indicate that FKHR and AFX, mammalian homologues of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. Here we describe the discovery of a novel AFX isoform, which we designated AFX zeta, in which the first 16 amino acids of the forkhead domain are not present. PCR analysis showed that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFX zeta induced reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, and G6Pase promoters. AFX zeta-mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression resulted in phosphorylation of AFX zeta. Furthermore, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an AMP-activated protein kinase activator, repressed AFX zeta-dependent reporter activation. Taken together, these findings suggest that AFX zeta is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway.