Nuclear receptors regulate metabolic pathways in response to changes in the environment by appropriate alterations in gene expression of key metabolic enzymes. Here, a computational search approach based on iteratively built hidden Markov models of nuclear receptors was used to identify a human nuclear receptor, termed hPAR, that is expressed in liver and intestines. hPAR was found to be efficiently activated by pregnanes and by clinically used drugs including rifampicin, an antibiotic known to selectively induce human but not murine CYP3A expression. The CYP3A drugmetabolizing enzymes are expressed in gut and liver in response to environmental chemicals and clinically used drugs. Interestingly, hPAR is not activated by pregnenolone 16␣-carbonitrile, which is a potent inducer of murine CYP3A genes and an activator of the mouse receptor PXR.1. Furthermore, hPAR was found to bind to and trans-activate through a conserved regulatory sequence present in human but not murine CYP3A genes. These results provide evidence that hPAR and PXR.1 may represent orthologous genes from different species that have evolved to regulate overlapping target genes in response to pharmacologically distinct CYP3A activators, and have potential implications for the in vitro identification of drug interactions important to humans.The recent progress made in determining the full genomic sequences of model organisms as well as the rapid accumulation of sequence data from the human genome has opened up new possibilities to determine the functional organization of genomes by computational approaches (1). Multiple alignments of members of different protein families followed by homology searching are powerful methods to infer gene function from sequence data as well as to identify novel genes within a given gene family. Hidden Markov models (HMMs) are a general statistical modeling technique that can be used as formal, fully probabilistic forms of sequence profiles (2, 3), describing the consensus of a set of sequences. This approach toward the identification and functional characterization of novel genes is particularly amenable to evolutionary conserved gene families consisting of a large number of orthologs and paralogs. The nuclear receptors constitute one such large gene family that is structurally and functionally conserved and represented within different metazoan phylae from cnidarians to vertebrates (4). These receptors are conditionally regulated transcription factors that exert their effects by interacting with small lipophilic ligands followed by sequence-specific binding of the receptor to DNA sequences called hormone response elements (HREs). Binding of the receptor to DNA results in changes in gene expression of specific target genes (5).Steroid hormones were the first group of small, lipophilic molecules identified as nuclear receptor ligands. Today, the number of substances known to regulate the activity of this group of receptors is represented by a large and chemically diverse group of molecules including retinoids, vi...

The nuclear receptor heterodimers of liver X receptor (LXR) and retinoid X receptor (RXR) are key transcriptional regulators of genes involved in lipid homeostasis and in¯ammation. We report the crystal structure of the ligand-binding domains (LBDs) of LXRa and RXRb complexed to the synthetic LXR agonist T-0901317 and the RXR agonist methoprene acid (Protein Data Base entry 1UHL). Both LBDs are in agonist conformation with GRIP-1 peptides bound at the coactivator binding sites. T-0901317 occupies the center of the LXR ligand-binding pocket and its hydroxyl head group interacts with H421 and W443, residues identi®ed by mutational analysis as critical for ligand-induced transcriptional activation by T-0901317 and various endogenous oxysterols. The topography of the pocket suggests a common anchoring of these oxysterols via their 22-, 24-or 27-hydroxyl group to H421 and W443. Polyunsaturated fatty acids act as LXR antagonists and an E267A mutation was found to enhance their transcriptional inhibition. The present structure provides a powerful tool for the design of novel modulators that can be used to characterize further the physiological functions of the LXR± RXR heterodimer.

The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPAR␥ isoform in glucose metabolism is illustrated by the fact that antidiabetic thiazolidinediones have been shown to be bona fide PPAR␥ ligands. Here we report the crystal structure of apo-PPAR␥ ligand binding domain (LBD) determined to 2.9-Å resolution. Although the structure of apo-PPAR␥-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPAR␥ LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPAR␥-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the -loop is extended in PPAR␥ and contains additional structural elements. Taken together, the apo-PPAR␥-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPAR␥ as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.

An approach to produce 13C- and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40-200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C(6)-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C(6)-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purifies IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20-40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.