Pharmacophore analysis of the nuclear oxysterol receptor LXRalpha.

A cell-free assay was developed for the orphan nuclear receptor LXRalpha that measures the ligand-dependent recruitment of a peptide from the steroid receptor coactivator 1 (SRC1) to the nuclear receptor. Using this ligand-sensing assay (LiSA), the structural requirements for activation of the receptor by oxysterols and related compounds were studied. The minimal pharmacophore for receptor activation was shown to be a sterol with a hydrogen bond acceptor at C24. 24(S),25-Epoxycholesterol (1), which meets this criterion, is among the most efficacious of the oxysterols and is an attractive candidate as the LXRalpha natural hormone. Cholenic acid dimethylamide (14) showed increased efficacy compared to 1, whereas the unnatural oxysterol 22(S)-hydroxycholesterol (4) was shown to be an antagonist of 1 in the LiSA. The structural requirements for SRC1 recruitment in the LiSA correlated with the transcriptional activity of compounds in a cell-based reporter assay employing LXRalpha-GAL4 chimeric receptors. Site-directed mutagenesis identified Trp(443) as an amino acid critical for activation of LXRalpha by oxysterol ligands. This information was combined with the structure-activity relationship developed from the LiSA to develop a 3D homology model of LXRalpha. This model may aid the design of synthetic drugs targeted at this transcriptional regulator of cholesterol homeostasis.

Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors.

The nuclear receptor PPARgamma/RXRalpha heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARgamma and RXRalpha LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARgamma agonist rosiglitazone or GI262570, and coactivator peptides. The PPARgamma/RXRalpha heterodimer is asymmetric, with each LBD deviated approximately 10 degrees from the C2 symmetry, allowing the PPARgamma AF-2 helix to interact with helices 7 and 10 of RXRalpha. The heterodimer interface is composed of conserved motifs in PPARgamma and RXRalpha that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARgamma/RXRbeta heterodimer by 9cRA.

Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands.

Xenobiotics induce the transcription of cytochromes P450 (CYPs) 2B and 3A through the constitutive androstane receptor (CAR; NR1I3) and pregnane X receptor (PXR; NR1I2), respectively. In this report, we have systematically compared a series of xenobiotics and natural steroids for their effects on mouse and human CAR and PXR. Our results demonstrate dual regulation of PXR and CAR by a subset of compounds that affect CYP expression. Moreover, there are marked pharmacological differences between the mouse (m) and human (h) orthologs of both CAR and PXR. For example, the planar hydrocarbon 1, 4-bis[2-(3,5-dichloropyridyl-oxy)]benzene activates mCAR and hPXR but has little or no activity on hCAR and mPXR. In contrast, the CAR deactivator androstanol activates both mouse and human PXR. Similarly, the PXR activator clotrimazole is a potent deactivator of hCAR. Using radioligand binding and fluorescence resonance energy transfer assays, we demonstrate that several of the compounds that regulate mouse and human CAR, including natural steroids, bind directly to the receptors. Our results suggest that CAR, like PXR, is a steroid receptor that is capable of recognizing structurally diverse compounds. Moreover, our findings underscore the complexity in the physiologic response to xenobiotics.

Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins.

Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.

Bile acids: natural ligands for an orphan nuclear receptor.

Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.

Overexpression and characterization of the human mitochondrial and cytosolic branched-chain aminotransferases.

We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.

Cloning of the rat and human mitochondrial branched chain aminotransferases (BCATm).

The rat and human mitochondrial branched chain aminotransferase (BCAT(m)) cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCAT(m) is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCAT(m) gene resides on chromosome 19 and the human cytosolic enzyme (BCAT(c)) gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed.

Cloning and expression of the mammalian cytosolic branched chain aminotransferase isoenzyme.

The cDNA for the rat cytosolic branched chain aminotransferase (BCATc) has been cloned. The BCATc cDNA encodes a polypeptide of 410 amino acids with a calculated molecular mass of 46.0 kDa. By Northern blot analysis, BCATc message of approximately 2.7 kilobases was readily detected in rat brain, but was absent from liver, a rat hepatoma cell line, kidney, and skeletal muscle. When expressed in COS-1 cells, the enzyme is immunologically indistinguishable from the native enzyme found in rat brain cytosol. Comparison of the rat BCATc sequence with available data bases identified the Escherichia coli (and Salmonella typhimurium) branched chain aminotransferase (BCAT) and revealed a Haemophilus influenzae BCAT, a yeast BCAT, which is hypothesized to be a mitochondrial form of the enzyme, and the murine BCATc (protein ECA39). Calculated molecular masses for the complete proteins are 33.9 kDa, 37.9 kDa, 42.9 kDa, and 43.6 kDa, respectively. The rat BCATc sequence was 84% identical with murine BCATc, 45% identical with yeast, 33% identical with H. influenzae, 27% identical with the E. coli and S. typhimurium BCAT, and 22% identical with the evolutionary related D-amino acid aminotransferase (D-AAT) (Tanizawa, K., Asano, S., Masu, Y., Kuramitsu, S., Kagamiyama, H., Tanaka, H., and Soda, K. (1989) J. Biol. Chem. 264, 2450-2454). Amino acid sequence alignment of BCATc with D-AAT suggests that the folding pattern of the overlapping mammalian BCATc sequence is similar to that of D-AAT and indicates that orientation of the pyridoxal phosphate cofactor in the active site of the eukaryotic BCAT is the same as in D-AAT. Thus, BCAT are the only eukaryotic aminotransferases to abstract and replace the proton on the re face of the pyridoxal phosphate cofactor. Finally, requirements for recognition of substrate L-amino acid and alpha-carboxylate binding are discussed.