Structure-Based Approach for the Study of Estrogen Receptor Binding Affinity and Subtype Selectivity

Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpa (hER alpha) and beta (hER beta). Because the levels and relative proportion of hER alpha and hER beta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hER alpha and hER beta. Significant statistical coefficients were obtained (hER alpha, q(2) = 0.76; hER beta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hER alpha and hER beta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design or novel hER modulators with improved selectivity.

Mapping the Intramolecular Vibrational Energy Flow in Proteins Reveals Functionally Important Residues

Unveiling the mechanisms of energy relaxation in biomolecules is key to our understanding of protein stability, allostery, intramolecular signaling, and long-lasting quantum coherence phenomena at ambient temperatures. Yet, the relationship between the pathways of energy transfer and the functional role of the residues involved remains largely unknown. Here, we develop a simulation method of mapping out residues that are highly efficient in relaxing an initially localized excess vibrational energy and perform site-directed mutagenesis functional assays to assess the relevance of these residues to protein function. We use the ligand binding domains of thyroid hormone receptor (TR) subtypes as a test case and find that conserved arginines, which are critical to TR transactivation function, are the most effective heat diffusers across the protein structure. These results suggest a hitherto unsuspected connection between a residue`s ability to mediate intramolecular vibrational energy redistribution and its functional relevance.

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring lambda 6 Light-Chain Fibrillogenesis

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda 6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda 6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wildtype protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda 6 LCs. (C) 2009 Elsevier Ltd. All rights reserved.

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics Simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics Simulations of the TR alpha and TR beta LBDs in the absence and in the presence of the natural ligand Triac. The Simulations Show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary Structure elements, while the Structure remains essentially compact, resembling a molten globule state. This differs From most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TR alpha and TR beta Subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our Simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H 11, and the interaction of the region between H I and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.

Adenosine Modulatesa alpha(2)-Adrenergic Receptors within Specific Subnuclei of the Nucleus Tractus Solitarius in Normotensive and Spontaneously Hypertensive Rats

Adenosine Is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A, receptors (A(1R)) on alpha(2)-adrenoceptors (Adr(2R)) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (B(max), K(d)) In the presence of 3 different concentrations of N(6)-cyclopentyladenosine (CPA), an A(1R) agonist. Neuronal culture confirmed our radioautographic results. [(3)H]RX821002, an Adr(2R) antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in B(max) values (21%) Induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by I nmol/L (increased B(max), 17%; decreased K(d), 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an Increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an AIR antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A(1R) that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important In understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension. (Hypertens Res 2008; 31: 2177-2186)

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

A novel physiological property of snake bradykinin-potentiating peptides-Reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [subtypes. BPP-10c did not induce receptor-mediated ion flux, nor potentiated carbamoylcholine-provoked receptor activity as determined by whole-cell recording. This peptide, however, alleviated MK-801-induced inhibition of nicotinic acetylcholine receptor activity. Although more data are needed for understanding the mechanism of the BPP-10c effect on nicotinic receptor activity and its relationship with the anti-hypertensive activity, this work reveals possible therapeutic applications for BPP-10c in establishing normal acetylcholine receptor activity. (C) 2008 Elsevier Inc. All rights reserved.

Estrogen receptor: Structural differences and potential implications on selectivity examined by the GRID/CPCA approach

Selective Estrogen Receptor Modulators ( SERMs) have been developed, but the selectivity towards the subtypes ( ER or ER is not well understood. Based on three-dimensional structural properties of ligand binding domains, a model that takes into account this aspect was developed via molecular interaction fields and consensus principal component analysis (GRID/CPCA).