The molecular mechanisms underlying the ERα-36-mediated signaling in breast cancer.

Alterations in estrogen-mediated cellular signaling have largely been implicated in the pathogenesis of breast cancer. Here, we investigated the signaling regulation of a splice variant of the estrogen receptor, namely estrogen receptor (ERα-36), associated with a poor prognosis in breast cancers. Coupling in vitro and in vivo approaches we determined the precise sequential molecular events of a new estrogen signaling network in an ERα-negative cell line and in an original patient-derived xenograft. After estrogen treatment, ERα-36 rapidly associates with Src at the level of the plasma membrane, initiating downstream cascades, including MEK1/ERK activation and paxillin phosphorylation on S126, which in turn triggers a higher expression of cyclin D1. Of note, the direct binding of ERα-36 to ERK2 prevents its dephosphorylation by MKP3 and enhances the downstream signaling. These findings improve our understanding of the regulation of non-genomic estrogen signaling and open new avenues for personalized therapeutic approaches targeting Src or MEK in ERα-36-positive patients.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands.

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1alpha,25(OH)(2)D(3) and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17beta-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Effects of ligand binding on the association properties and conformation in solution of retinoic acid receptors RXR and RAR.

In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-trans retinoic acid (RA), are involved in the regulation of several essential physiological processes. Their pleiotropic physiological effects are mediated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuclear hormone receptors. Hormone binding to the structurally conserved ligand-binding domain (LBD) of these receptors triggers a conformational change that principally affects the conserved C-terminal transactivation helix H12 involved in transcriptional activation. We report an extensive biophysical solution study of RAR alpha, RXR alpha LBDs and their corresponding RXR alpha/RAR alpha LBD heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scattering (SAXS and SANS) and ab initio three-dimensional shape reconstruction at low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthermore we demonstrate the effects of 9-cisRA and all-transRA binding on the association properties and conformations of RXR alpha and RAR alpha LBDs in solution. The present study shows that in solution RAR alpha LBD behaves as a monomer in both unliganded and liganded forms. It confirms the existence in solution of a ligand-induced conformational change towards a more compact form of the LBD. It also confirms the stability of the predicted RXR alpha/RAR alpha LBD heterodimers in solution. SAS measurements performed on three different types of RXR alpha/RAR alpha LBD heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the existence of three conformational states depending on the progressive binding of RA stereoisomers on RAR alpha and RXR alpha LBD subunits in the heterodimeric context. These results suggest that the subunits are structurally independent within the heterodimers. Our study also underlines the particular behaviour of RXR alpha LBD. In solution unliganded RXR alpha LBD is observed as two species that are unambiguously identified as homotetramers and homodimers. Molecular modelling combined with SAS data analysis allows us to propose a structural model for this autorepressed apo-tetramer. In contrast to the monomeric state observed in the crystal structure, our data show that in solution active holo-RXR alpha LBD bound to 9-cisRA is a homodimer regardless of the protein concentration. This study demonstrates the crucial role of ligands in the regulation of homodimeric versus heterodimeric association state of RXR in the NR signalling pathways.

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor.

Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild-type is not well ordered.

Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects.

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.

Crystal structure of the human RXRalpha ligand-binding domain bound to its natural ligand: 9-cis retinoic acid.

The pleiotropic effects of active retinoids are transduced by their cognate nuclear receptors, retinoid X receptors (RXRs) and retinoic acid receptors (RARs), which act as transcriptional regulators activated by two stereoisomers of retinoic acid (RA): 9-cis RA (9-cRA) and all-trans RA (a-tRA). Among nuclear receptors, RXR occupies a central position and plays a crucial role in many intracellular signalling pathways as a ubiquitous heterodimerization partner with numerous other members of this superfamily. Whereas RARs bind both isomers, RXRs exclusively bind 9-cRA. The crystal structure of the ligand-binding domain (LBD) of human RXRalpha bound to 9-cRA reveals the molecular basis of this ligand selectivity and allows a comparison of both apo and holo forms of the same nuclear receptor. In the crystal, the receptor is monomeric and exhibits a canonical agonist conformation without direct contacts between the ligand and the transactivation helix H12. Comparison with the unliganded RXRalpha LBD structure reveals the molecular mechanisms of ligand-induced conformational changes and allows us to describe at the atomic level how these changes generate the proper protein interface involved in nuclear receptor-coactivator interaction.

The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand.

The action of 1 alpha, 25-dihydroxyvitamin D3 is mediated by its nuclear receptor (VDR), a ligand-dependent transcription regulator. We report the 1.8 A resolution crystal structure of the complex between a VDR ligand-binding domain (LBD) construct lacking the highly variable VDR-specific insertion domain and vitamin D. The construct exhibits the same binding affinity for vitamin D and transactivation ability as the wild-type protein, showing that the N-terminal part of the LBD is essential for its structural and functional integrity while the large insertion peptide is dispensable. The structure reveals the active conformation of the bound ligand and allows understanding of the different binding properties of some synthetic analogs.

Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies.

We have expressed recombinant human vitamin D receptor and its ligand-binding domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its ligand-binding domain were present as monomers in solution. The purified vitamin D receptor and its ligand-binding domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the ligand-binding domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its ligand-binding domain in insect cells and purify them for functional and structural studies.

Dependence of the lytic activity of the N-terminal domain of human perforin on membrane lipid composition--implications for T-cell self-preservation.

The kinetics and thermodynamics of pore formation by the 22-residue N-terminal domain of human perforin-(1-22)-peptide have been studied for a variety of model phospholipid membranes. Peptide binding and aggregation, and cell lysis were monitored through the change in the fluorescence of Trp, or vesicle-encapsulated carboxyfluorescein, respectively. Peptide binding was analyzed in terms of a model that incorporates non-ideal interactions and aggregation in a membrane bilayer. The minimum number of peptide monomers required to form an active pore averaged from four to six, according to the lipid composition of the vesicle. This combined kinetic and thermodynamic approach has provided quantitative information that allows a direct comparison of the binding behavior of the perforin-(1-22)-peptide in different lipid vesicles and affords molecular insight on the factors controlling pore formation. Pore formation is most favorable in thinner membranes with low melting temperatures. No significant difference in activity is observed for different zwitterionic headgroups. Rather, the gel state of the lipid chain, which diminishes the incorporation and aggregation of the perforin-(1-22)-peptide shows the strongest influence. This effect is observed in both the thermodynamic (incorporation isotherm) and kinetic (carboxyfluorescein release) studies. Insertion and aggregation are more facile in membranes with less densely packed lipids. The dependence of pore-forming activity on lipid composition provides important clues to understanding the self-protection mechanism employed by cytotoxic T lymphocytes (CTL) against perforin-mediated lysis.

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.

Negative cooperativity exhibited by the lytic amino-terminal domain of human perforin: implications for perforin-mediated cell lysis.

BACKGROUND: Cytolytic effector cells of the immune system recognize and lyse cells that carry non-self epitopes. One mechanism of cell lysis involves release of a 67-kDa pore-forming protein, perforin. The amino-terminal domain of perforin (>/= 19 residues) can account for most of the lysis activity, by a mechanism that is similar to that of holoperforin. Detailed mechanistic studies of this domain should yield useful insight into the factors underlying perforin activity in vivo. RESULTS: The mechanism of pore formation by the 22-residue amino-terminal domain of perforin was studied by kinetic and thermodynamic methods. Approximately 4 +/- 1 peptide monomers form an active pore by a mechanism that displays negative cooperativity. CONCLUSIONS: A negatively-regulated aggregation mechanism is likely to be common for pore-forming peptides. The positively-charged domain B of perforin (residues 7- 15) may mediate cooperativity through electrostatic interactions. Such a mechanism limits the number of protein molecules that are committed to any particular channel. This data supports smaller pores as the physiologically relevant aggregate, rather than the larger ring sizes identified by electron microscopy at higher, non-biological concentrations.

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.

Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E.