3,5-T2 is an alternative ligand for the thyroid hormone receptor ß1.

Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T(2)), a naturally occurring iodothyronine that results from T(3) outer-ring deiodination, is an alternative ligand for thyroid hormone receptor ß1 (TRß1). In tilapia, 2 TRß isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T(2) activates the human and the long tilapia TRß1 isoform, but not the short one. A chimeric human TRß1 (hTRß1) that contained the 9-amino-acid insert showed no response to T(2), suggesting that the conformation of the hTRß1 naturally allows T(2) binding and that other regions of the receptor are implicated in TR activation by T(2). Indeed, further analysis showed that the N terminus is essential for T(2)-mediated transactivation but not for that by T(3) in the long and hTRß1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T(2)-mediated TRß1 binding and activation, mRNA expression and its regulation by T(2) and T(3) was evaluated for both isoforms. Our data show that long TRß1expression is 10(6)-fold higher than that of the short isoform, and T(3) and T(2) differentially regulate the expression of these 2 TRß1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors.

X-ray structure of the orphan nuclear receptor RORbeta ligand-binding domain in the active conformation.

The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.

A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity.

FMOC-L-Leucine (F-L-Leu) is a chemically distinct PPARgamma ligand. Two molecules of F-L-Leu bind to the ligand binding domain of a single PPARgamma molecule, making its mode of receptor interaction distinct from that of other nuclear receptor ligands. F-L-Leu induces a particular allosteric configuration of PPARgamma, resulting in differential cofactor recruitment and translating in distinct pharmacological properties. F-L-Leu activates PPARgamma with a lower potency, but a similar maximal efficacy, than rosiglitazone. The particular PPARgamma configuration induced by F-L-Leu leads to a modified pattern of target gene activation. F-L-Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant, and in diabetic db/db mice, yet it has a lower adipogenic activity. These biological effects suggest that F-L-Leu is a selective PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

Binding of ligands and activation of transcription by nuclear receptors.

Nuclear receptors (NRs) form a superfamily of ligand-inducible transcription factors composed of several domains. Recent structural studies focused on domain E, which harbors the ligand-binding site and the ligand-dependent transcription activation function AF-2. Structures of single representatives in an increasing number of various complexes as well as new structures of further NRs addressed issues such as discrimination of ligands, superagonism, isotype specificity, and partial agonism. Until today, one unique transcriptionally active form of domain E was determined; however, divergent tertiary structures of apo-forms and transcriptionally inactive forms are known. Thus, recent results link the transformation of NRs upon ligand binding to principles of protein folding. Furthermore, the ensemble of NR structures, including those of DNA-binding domains, provides one of the foundations for the understanding of interactions with transcription intermediary factors up to the characterization of the link between NR complexes and the basal transcriptional machinery at the structural level.

The pleiotropic functions of peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for synthetic compounds that induce peroxisome proliferation. PPARgamma is the best characterized of the PPARs. The heterodimer of PPARgamma with the retinoid X receptor (RXR) plays a crucial role in adipogenesis and insulin sensitization. The RXR/PPARgamma heterodimer furthermore has been reported to have important immunomodulatory activities and to affect cell proliferation/differentiation pathways in various malignancies. PPARgamma is activated by a number of naturally occurring fatty acid derivatives and by several synthetic compounds, including the thiazolidinediones and L-tyrosine-based insulin sensitizers. This review gives an overview of the pleiotropic functions of PPARgamma and discusses the wide-ranging medical implications that modulation of PPARgamma activity might have for various diseases, ranging from obesity and type 2 diabetes to cancer and inflammation.

Structural studies on nuclear receptors.

Nuclear receptors are DNA-binding factors which regulate the transcription of sets of specific genes in response to cognate ligands, usually small lipophilic molecules, thus controlling numerous physiological events in development, procreation, homeostasis, and cellular life. Their ligand-dependent activity makes nuclear receptors obvious targets for drug design in many therapeutic areas. Crystallographic studies have revealed the structure of isolated domains but not, yet, of a whole protein, probably due to an intrinsic flexibility at work in nuclear receptor action. The structure of DNA-binding domain dimers in complex with an oligonucleotide has brought insights into how nuclear receptors recognize and bind to their target sequences ('response elements'). The structure of several ligand-binding domains in different ligation states has provided evidence for a ligand-dependent transcriptional switch and a molecular basis for the mode of action of agonists and antagonists.

Structure-function analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains.

Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13delta1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.

Retinoic acid receptor: a simulation analysis of retinoic acid binding and the resulting conformational changes.

The binding/escape mechanism of all- trans retinoic acid with respect to the ligand-binding domain of the nuclear receptor RARgamma has been studied by molecular dynamic simulations. The entry/exit channel was shown to be on the side of the activation helix by the use of multiple copy dynamics. Three independent minimum energy paths from the liganded structure to a model for the unliganded structure were calculated with the conjugate peak refinement method. Ligand escape takes place in the early steps of the transition during rearrangement of the binding pocket; the latter involves inward motion of the beta-sheet and outward motions of the Omega-loop and helix H6. The correlated rearrangements involved in the escape phase are similar and occur in the same order for the different paths. After the escape phase, the conformational changes affect primarily the C-terminal helices H11-H12 and the Omega-loop. The three paths are significantly different for this reorganization phase and reveal a multiplicity of possibilities, in agreement with the idea that the apo state is structurally less constrained. The present calculations extend the crystallographic results, confirming the "mouse trap" mechanism and stressing the importance of the helix H3 conformation and of the contacts between the Omega-loop and helices H11 and H6. They are in good agreement with known mutants and point to other functionally important residues, especially in helices H3 and H11, suggesting mutations that may affect the ligand-binding function and the associated conformational changes.

Purification of the human RARgamma ligand-binding domain and crystallization of its complex with all-trans retinoic acid.

A 28-kDa fragment (residues 178-423) of the human retinoic acid receptor gamma, hRARgamma D3E, encompassing the ligand-binding domain (LBD) was overproduced in Escherichia coli and purified as a monomer to more than 95% purity and homogeneity. The Kd for all-trans retinoic acid binding was 0.6 +/- 0.1 nM. Crystals of the LBD complexed with all-trans retinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method. They diffract to 2.0 A using a synchrotron radiation (lambda=0.91 A) and belong to the tetragonal space group P4(1)2(1)2 with unit cell parameters a=b=60.6 A and c=155.3 A, one monomer per asymmetric unit, a solvent content of ca. 33%, and a Vm value of approximately 2 A3/dalton.

Effects of ozone on yield, growth, and root starch concentrations of two alfalfa (Medicago sativ A L.) cultivars.

Ozone (O3) is considered to be a major air pollutant that affects the yield of several sensitive crop species. Its concentration may reach phytotoxic levels several times during the growing season in Eastern Canada. This study was initiated to evaluate the O3 effects on alfalfa, a major crop species. The objective was to compare the yield and growth parameters of the main alfalfa cultivar used in Québec, Apica, to a cultivar more tolerant to O3, Team. Effects on root starch concentrations were also examined as this parameter is an important indicator of alfalfa perennity. The results obtained have shown that the forage yield of Apica was more reduced by O3 during two growing seasons than the yield of Team. For O3 concentrations of 20 to 40 nl liter(-1), yield reductions were 14-26% for Apica and 0-20% for Team. Whereas Apica could be considered more susceptible to O3 than Team, the latter has shown contrasting responses from year to year. This fact suggests that the mechanisms involved in O3-tolerance could be modulated by environmental conditions. At low O3 levels, Apica has shown reduced root growth in terms of dry matter and length. However, contrary to the current hypothesis that O3 would affect more root than shoot growth, we were unable to show a consistent alteration of the biomass allocation between the two. Ozone seems to reduce globally the growth of the whole plants. The greater O3-tolerance of Team could partly be associated to its capacity to maintain more leaves, to delay their senescence, or to keep a larger leaf:stem ratio under increasing levels of O3. At the end of the two growing seasons, the amount of starch reserves stored below ground was shown to be reduced by the current O3 levels. This reduction was mainly associated with a decrease in root biomass under O3 stress. This result support the hypothesis that O3 may accelerate alfalfa decline under field conditions.

Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP.

A connection between transcription and DNA repair was demonstrated previously through the characterization of TFIIH. Using filter binding as well as in vitro transcription challenge competition assays, we now show that the promoter recognition factor TATA box-binding protein (TBP)/TFIID binds selectively to and is sequestered by cisplatin- or UV-damaged DNA, either alone or in the context of a larger protein complex including TFIIH. Computer-assisted 3D structural analysis reveals a remarkable similarity between the structure of the TATA box as found in its TBP complex and that of either platinated or UV-damaged oligonucleotides. Thus, cisplatin-treated or UV-irradiated DNA could be used as a competing binding site which may lure TBP/TFIID away from its normal promoter sequence, partially explaining the phenomenon of DNA damage-induced inhibition of RNA synthesis. Consistent with an involvement of damaged DNA-specific binding of TBP in inhibiting transcription, we find that microinjection of additional TBP in living human fibroblasts alleviates the reduction in RNA synthesis after UV irradiation. Future anticancer drugs could be designed with the consideration of lesion recognition by TBP and their ability to reduce transcription.

Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes.

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177-185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.

A canonical structure for the ligand-binding domain of nuclear receptors.

The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXR alpha and the liganded RAR gamma LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specific signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXR alpha and glucocorticoid receptor ligand-binding sites according to the RAR gamma holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RAR gamma holo- and inactive RXR alpha apo-LBD structures.

Crystal structure of the RAR-gamma ligand-binding domain bound to all-trans retinoic acid.

The 2.0-A crystal structure of the ligand-binding domain (LBD) of the human retinoic acid receptor (RAR)-gamma bound to all-trans retinoic acid reveals the ligand-binding interactions and suggests an electrostatic guidance mechanism. The overall fold is similar to that of the human RXR-alpha apo-LBD, except for the carboxy-terminal part which folds back towards the LBD core, contributing to the hydrophobic ligand pocket and 'sealing' its entry site. We propose a 'mouse trap' mechanism whereby a ligand-induced conformational transition repositions the amphipathic alpha-helix of the AF-2 activating domain and forms a transcriptionally active receptor.

High affinity of ergopeptides for cytochromes P450 3A. Importance of their peptide moiety for P450 recognition and hydroxylation of bromocriptine.

The interaction between rat and human liver cytochromes P450 with a series of lysergic acid derivatives and ergopeptide alkaloids was studied by difference visible spectroscopy. Ergopeptides, like bromocriptine, ergocryptine and dihydroergotamine, strongly interacted with rat liver microsomes with the appearance of a difference spectrum which is characteristic of their binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P450s 3A in the microsomes and was at its maximum in dexamethasone-treated rat microsomes. All the ergopeptides studied exhibited a high affinity for rat P450s 3A (Ks around 1 microM), although lysergic acid derivatives not bearing the tripeptide moiety failed to give significant interactions with these P450s. A cyclic azatripeptide exhibiting a structure very similar to that of the tripeptide moiety of ergopeptides also interacted with P450s 3A with appearance of an intense type I difference spectrum. Very similar results were observed with two allelic forms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeast. In both cases all the ergopeptides studied showed high affinities for the P450s (Ks 0.6-2.2 microM) and an intense shift from the low-spin to the high-spin state upon substrate binding (60-100% spin shift). Lysergic acid derivatives not bearing the tripeptide group of ergopeptides also completely failed to interact with P450s 3A4. Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of bromocriptine, which occurs at the level of its tripeptide moiety. Human liver microsomes as well as P450 NF25 and P450 hPCN1 also exhibited a high activity for bromocriptine hydroxylation at this level. These results show that ergopeptides exhibit a particularly high affinity for P450s of the 3A subfamily. The tripeptide moiety of ergopeptides is essential for their recognition by P450s 3A and binds at a site close to P450 heme, producing type-I difference spectra. Accordingly, at least one of the studied ergopeptides, bromocriptine, is hydroxylated by P450s 3A at the proline ring of the cyclopeptide moiety. As cyclosporine is known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.

Expression in yeast of three allelic cDNAs coding for human liver P-450 3A4. Different stabilities, binding properties and catalytic activities of the yeast-produced enzymes.

Three natural allelic cDNAs coding for P-450 3A4, the major form in human liver, namely NF25, NF10 and hPCN1, have been expressed in Saccharomyces cerevisiae. NF25 and hPCN1 were functionally expressed in yeast microsomes, yielding proteins with an absorption maximum at 448 nm in the CO-reduced difference spectrum. Some catalytic activities and substrate binding properties of P-450 NF25 and P-450 hPCN1 in yeast microsomes have been compared; no striking difference was found, showing that the two point substitutions between their amino-acid sequences (Trp392 and Thr431 in P-450 NF25 are replaced by Val392 and Ile431 in P-450 hPCN1) have no significant effect on the functional properties of these two variants. By contrast, P-450 NF10, which differs from P-450 NF25 by a one-amino-acid deletion (Ile224 replacing Thr224-Val225), was produced as a denatured form, as revealed by an absorption maximum at 420 nm, and was not catalytically active. This suggests that the deletion prevents the correct folding of the protein. The results of this study show that P-450 NF25 and P-450 hPCN1 are two roughly equivalent, functionally active variants of P-450 3A4, but that P-450 NF10 is a defective, unstable gene product that could arise from an alternative mRNA splicing. This could contribute to the large variations reported for nifedipine oxidation, a typical P-450 3A4 activity, in human liver.

Recombinant yeast in drug metabolism.

The usefulness of cDNA-directed expression of human hepatic P450s in yeast for the in vitro study of drug metabolism is emphasized. The major advantages of yeast expression are: (i) relatively high yields of heterologous P450 (approximately 5-10 nmol/l of culture medium) can be obtained; (ii) the expressed P450s are directly active in yeast microsomes, allowing the determination of specific catalytic activities of individual isoforms, which is a prerequisite for the prediction of metabolic pathways for new drug candidates; (iii) transformed yeast microsomes can also be used to study the specific affinity of individual P450s for various substrates and the formation of P450-metabolite complexes by difference visible spectroscopy; such studies can help to predict drug interactions. The advantages of expression in yeast with respect to biochemical studies of drug metabolism are illustrated with data about P450 NF25 (P450 3A4), the major form of human liver. Expressed P450 NF25 is obtained in a functionally active state, and some specific catalytic activities observed in liver microsomes could be reproduced directly with transformed yeast microsomes. The use of genomically modified yeast strains coexpressing human cytochrome b5 and/or overexpressing yeast P450-reductase allowed us to optimize these catalytic activities. In particular, this coexpression system was useful in the study of the in vitro formation of a P450 NF25 Fe(II)-RNO complex. Such inhibitory complexes have been implied in numerous drug interactions involving P450 3A4.

Particular ability of liver P450s3A to catalyze the oxidation of N omega-hydroxyarginine to citrulline and nitrogen oxides and occurrence in no synthases of a sequence very similar to the heme-binding sequence in P450s.

Liver microsomes from rats pretreated with various inducers of P450 isoforms exhibit very different abilities to catalyze the oxidation of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides. Treatment of rats with dexamethasone, a classical inducer of P450 3A, leads to a spectacular 7-fold increase of the activity found for untreated rats, while induction by phenobarbital causes a much lower increase of this activity and induction by 3-methylcholanthrene or clofibrate decreases it. Specific inhibitors of P450s3A as troleandomycin and dihydroergotamine strongly inhibit NOHA oxidation whereas metyrapone, an inhibitor of other P450 subfamilies, was without effect. These data show the particular ability of P450s of the 3A subfamily to catalyze the second step of the oxidation of L-arginine by NO synthases (NOS). This analogy between NOSs and P450s3A is further substantiated by a protein sequence comparison which shows that a 9-amino acid segment present in all NOSs exhibits a strong similarity with the sequence mainly responsible for heme binding in P450s3A which is well conserved in all P450s. This segment contains all the structural factors which are thought to be crucial for heme binding in P450s.