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Anobel Tamrazi

Publications and source records attributed to Anobel Tamrazi.

8 recordsLinked to original sources
PubMed2005

Coactivator proteins as determinants of estrogen receptor structure and function: spectroscopic evidence for a novel coactivator-stabilized receptor conformation.

The direct regulation of gene transcription by nuclear receptors, such as the estrogen receptor (ER), involves not just ligand and DNA binding but the recruitment of coregulators. Typically, recruitment of p160 coactivator proteins to agonist-liganded ER is considered to be unidirectional, with ligand binding stabilizing an ER ligand binding domain (LBD) conformation that favors coactivator interaction. Using fluorophore-labeled ERalpha-LBDs, we present evidence for a pronounced stabilization of ER conformation that results from coactivator binding, manifest by decreased ER sensitivity to proteases and reduced conformational dynamics, as well as for the formation of a novel coactivator-stabilized (costabilized) receptor conformation, that can be conveniently monitored by the generation of an excimer emission from pyrene-labeled ERalpha-LBDs. This costabilized conformation may embody features required to support ER transcriptional activity. Different classes of coactivator proteins combine with estrogen agonists of different structure to elicit varying degrees of this receptor stabilization, and antagonists and coactivator binding inhibitors disfavor the costabilized conformation. Remarkably, high concentrations of coactivators engender this conformation even in apo- and antagonist-bound ERs (more so with selective ER modulators than with pure antagonists), providing an in vitro model for the development of resistance to hormone therapy in breast cancer.

Allosteric Site↗
PubMed2004

A proteomic microarray approach for exploring ligand-initiated nuclear hormone receptor pharmacology, receptor selectivity, and heterodimer functionality.

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.

Binding Sites↗
PubMed2004

Estrogen receptor microarrays: subtype-selective ligand binding.

We present the first example of a nuclear hormone receptor microarray, using for illustration the ligand-binding domains of the two estrogen receptors, ERalpha-LBD and ERbeta-LBD. The proteins are printed and allowed to attach to aldehyde slides; the efficiency of attachment depends on whether the LBD is liganded with agonists (low attachment) versus liganded with antagonists or unliganded (high attachment). This suggests that attachment is orientation specific and involves principally a single lysine residue. The attached ERs retain good ligand-binding activity that can be assessed using an estradiol-fluorophore conjugate, and specific and ER subtype-selective binding of ligands can be determined conveniently in competitive binding assays. This powerful new, high-throughput technique to study ligand binding to ER-LBDs can be extended to other nuclear hormone receptors and adapted to assay the recruitment of coregulator proteins.

Aldehydes↗
PubMed2004

Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling.

Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the bound ligand. Furthermore, it documents the complexity and uniqueness of the ligand-receptor structure, and highlights the fact that structural differences exist between the receptor bound with ligands of different pharmacological character that, nevertheless, produce similar crystal structures.

Amino Acid Substitution↗
PubMed2004

Design, synthesis, and in vitro biological evaluation of small molecule inhibitors of estrogen receptor alpha coactivator binding.

Nuclear receptors (NRs) complexed with agonist ligands activate transcription by recruiting coactivator protein complexes. In principle, one should be able to inhibit the transcriptional activity of the NRs by blocking this transcriptionally critical receptor-coactivator interaction directly, using an appropriately designed coactivator binding inhibitor (CBI). To guide our design of various classes of CBIs, we have used the crystal structure of an agonist-bound estrogen receptor (ER) ligand binding domain (LBD) complexed with a coactivator peptide containing the LXXLL signature motif bound to a hydrophobic groove on the surface of the LBD. One set of CBIs, based on an outside-in design approach, has various heterocyclic cores (triazenes, pyrimidines, trithianes, cyclohexanes) that mimic the tether sites of the three leucines on the peptide helix, onto which are appended leucine residue-like substituents. The other set, based on an inside-out approach, has a naphthalene core that mimics the two most deeply buried leucines, with substituents extending outward to mimic other features of the coactivator helical peptide. A fluorescence anisotropy-based coactivator competition assay was developed to measure the specific binding of these CBIs to the groove site on the ER-agonist complex with which coactivators interact; control ligand-binding assays assured that their interaction was not with the ligand binding pocket. The most effective CBIs were those from the pyrimidine family, the best binding with K(i) values of ca. 30 microM. The trithiane- and cyclohexane-based CBIs appear to be poor structural mimics, because of equatorial vs axial conformational constraints, and the triazene-based CBIs are also conformationally constrained by amine-substituent-to-ring resonance overlap, which is not the case with the higher affinity alkyl-substituted pyrimidines. The pyrimidine-based CBIs appear to be the first small molecule inhibitors of NR coactivator binding.

Amino Acid Motifs↗
PubMed2003

Molecular sensors of estrogen receptor conformations and dynamics.

The ligand-induced conformation of a nuclear receptor ligand-binding domain (LBD) is a principal factor leading to transcriptional activity and determining the pharmacological response. Using the estrogen receptor (ER) LBD-labeled site specifically with a fluorophore, we demonstrate that effects of ligand binding on the conformation and dynamics of this domain can be studied directly, in a quantitative and convenient fashion, by various fluorescence methods. Estrogen ligands of different pharmacological character-agonists, selective ER modulators (SERMs), and pure antagonists-each produce distinctive spectroscopic signatures, characteristic of the conformational or dynamic features of their ER-LBD complexes. We can directly follow the equilibrium of helix 12 positions through the degree of local fluorophore rotational freedom and receptor helicity near the C terminus of helix 11. We observe differences even between ligands within a specific pharmacological class, such as the SERMs raloxifene and trans-4-hydroxytamoxifen, highlighting the ability of these fluorescent receptor sensors to detect unique ER conformations induced even by closely related ligands, yet ones that produce distinctive biological activities in estrogen target cells. Fluorophore-labeled LBDs can serve as versatile molecular sensors predictive of ligand pharmacological character and should be broadly applicable to other members of the nuclear receptor superfamily.

Binding Sites↗
PubMed2002

Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate.

Nuclear receptors form strong dimers that are essential for their function as transcription factors, and it is thought that ligand binding can affect dimer stability. In this report, we describe convenient fluorescence resonance energy transfer (FRET)-based methods for measuring the thermodynamic and kinetic stability of dimers of the estrogen receptor-alpha ligand-binding domain (ERalpha-LBD). We have developed receptors that are chemically labeled with a single fluorophore in a site-specific manner. These fluorophore-labeled ERs are functional and can be used to measure directly the affinity and stability of ERalpha-LBD dimers. Our results indicate that unliganded ERalpha-LBDs exist as very stable dimers and that the dissociation rate of these dimers is slow (t(1/2)=39 +/- 3 min at 28 C) and is further slowed (< or =7-fold) by the addition of various ligands. Estrogen antagonists provide greater kinetic stabilization of the ER dimers than agonists. In addition, coactivator peptides containing the LXXLL motif selectively stabilize agonist-bound ERalpha-LBD dimers. These fluorescence-based assays for measuring the kinetic and thermodynamic stability of ER dimers provide a functional in vitro method for assessing the agonist or antagonist character of novel ligands.

Binding Sites↗