Ligand entry pathways in the ligand binding domain of PPARγ receptor

To address the question of ligand entry process, we report targeted molecular dynamics simulations of the entry of the flexible ionic ligand GW0072 in the ligand binding domain of the nuclear receptor PPARγ. Starting with the ligand outside the receptor the simulations led to a ligand docked inside the binding pocket resulting in a structure very close to the holo-form of the complex. The results showed that entry process is guided by hydrophobic interactions and that entry pathways are very similar to exit pathways. We suggest that TMD method may help in discriminating between ligands generated by in silico docking.     

Is the isolated ligand binding domain a good model of the domain in the native receptor?

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.     

Regio and stereospecific synthesis of 11β-substituted 19-norsteroids: Influence of 11β-substitution on progesterone receptor affinity — (1)

The convenient synthesis of a series of lie-substituted 17-ethynyl-17β-hydroxy-4, 9-estradien-3-ones and their corresponding estradiols is reported. Relative affinities for the progestin and estrogen receptors showed very specific interactions between the progesterone receptor and the unsaturated substituents in the 11β-position of the steroid.     

Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand,hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.     

Regulation of integrin αIIbβ3 ligand binding and signaling by the metal ion binding sites in the β I domain

The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.     

Analysis of exposed regions on the main extracellular domain of mouse acetylcholine receptor α subunit in live muscle cells by binding profiles of antipeptide antibodies

To study the structural organization of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) subunit in live muscle cells, we examined the native membrane-bound receptors in cultured mouse skeletal muscle cells for their ability to bind a panel of antibodies against uniform-sized overlapping synthetic peptides which collectively represent this entire domain. The binding profile indicated that the regions 23–49,78–126,146–174, and182–210 are accessible to binding with antibody. Residues23–49,78–126, and194–210 contain binding regions for-neurotoxin and some myasthenia gravis autoantibodies. A comparison of this binding profile with the profile obtained for membrane-boundTorpedo californica AChR in isolated membrane fractions showed some similarities as well as significant differences between the subunit organization in the isolated membrane fraction and that in the membrane of live muscle cells. Regions89–104 and158–174, which are exposed in the isolated membrane fraction, are also exposed in the live cell. On the other hand, regions23–49, and182–210, which are exposed in the live cell, are not accessible in the isolated membrane and, furthermore, the region1–16, which has marginal accessibility in the cell, becomes highly accessible in the membrane isolates. The exposed regions defined by this study may be the primary targets for the initial autoimmune attack on the receptors in experimental autoimmune myasthenia gravis.     

Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.     

Application of liquid chromatography–turbo ion spray tandem mass spectrometry for quantitative analysis of a potent motilin receptor agonist,EM574, and its metabolites in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the simultaneous determination of a new potent motilin receptor agonist as erythromycin derivative, EM574 (erythromycin derivative), and its three metabolites, M-IV, M-V and M-VI, in human plasma was developed. The internal standards (I.S.s) used were deuterated EM574, M-IV and M-V. For the quantitation of M-VI, deuterated M-V was used. The analytes and I.S. were extracted from plasma samples with diethyl ether at neutral pH. A turbo ion spray interface was used as the ion source of LC–MS–MS, and the analysis was performed in the selected reaction monitoring mode. The lower quantitation limits for all the analytes were 0.05 ng/ml when 0.2 ml of plasma was used, and the standard curves were linear in the range 0.05 to 20 ng/ml. The method was precise; the intra- and inter-day precisions of the method were not more than 19.8% for all the analytes. The accuracy of the method was good with the deviations between added and calculated concentrations of each analyte being typically within ±11.2%.     

β-Lactotensin derived from bovine β-lactoglobulin exhibits anxiolytic-like activity as an agonist for neurotensin NTS(2) receptor via activation of dopamine D(1) receptor in mice

β-Lactotensin (His-Ile-Arg-Leu) is a bioactive peptide derived from bovine milk β-lactoglobulin, acting as a natural agonist for neurotensin receptors. We found that β-lactotensin exhibited anxiolytic-like activity in an elevated plus-maze test after its intraperitoneal (i.p.) administration in mice. β-Lactotensin was also orally active. The anxiolytic-like activity of β-lactotensin after i.p. administration was blocked by levocabastine, an antagonist for the neurotensin NTS(2) receptor. β-Lactotensin had anxiolytic-like activity in wild-type but not Ntsr2-knockout mice. β-Lactotensin increased intracellular Ca(2+) flux in glial cells derived from wild-type mice but not Ntsr2 knockout mice. These results suggest that β-lactotensin acts as an NTS(2) receptor agonist having anxiolytic-like activity. The anxiolytic-like activity of β-lactotensin was also blocked by SCH23390 and SKF83566, antagonists for dopamine D(1) receptor, but not by raclopride, an antagonist for D(2) receptor. Taken together, β-lactotensin may exhibit anxiolytic-like activity via NTS(2) receptor followed by D(1) receptor.     

Synthesis and evaluation of 17α-E-20-(heteroaryl)norpregn-1,3,5(10),20 tetraene-3,17β-diols [17α-(heteroaryl)vinyl estradiols] as ligands for the estrogen receptor-α ligand binding domain (ERα-LBD)

A series of 17α-(heteroaryl)vinyl estradiols was prepared to evaluate the influence of heteroatom on the affinity and efficacy of estrogenic ligands for the estrogen receptor-alpha ligand binding domain (ERα-LBD). The products demonstrated reduced binding affinity compared to the parent 17α-E-phenyl vinyl estradiol, but the binding was relatively independent of the heteroatom. The greatest influence of the heteroatom was evident in the efficacy of the compounds as the thienyl derivatives 2f,g were more potent than either the pyridyl 2b-d or pyrimidinyl 2e analogs. The results suggest that a subtle interplay of interactions between the ligands and the receptor influences the biological response.