Evolutionary information helps understand distinctive features of the angiotensin II receptors AT1 and AT2 in amniota.

In vertebrates, the octopeptide angiotensin II (AngII) is an important in vivo regulator of the cardiovascular system. It acts mainly through two G protein-coupled receptors, AT1 and AT2. To better understand distinctive features of these receptors, we carried out a phylogenetic analysis that revealed a mirror evolution of AT1 and AT2, each one split into two clades, separating fish from terrestrial receptors. It also revealed that hallmark mutations occurred at, or near, the sodium binding site in both AT1 and AT2. Electrostatics computations and molecular dynamics simulations support maintained sodium binding to human AT1 with slow ingress from the extracellular side and an electrostatic component of the binding free energy around -3kT, to be compared to around -2kT for human AT2 and the δ opioid receptor. Comparison of the sodium binding modes in wild type and mutated AT1 and AT2 from humans and eels indicates that the allosteric control by sodium in both AT1 and AT2 evolved during the transition from fish to amniota. The unusual S7.46N mutation in AT1 is mirrored by a L3.36M mutation in AT2. In the presence of sodium, the N7.46 pattern in amniota AT1 stabilizes the inward orientation of N3.35 in the apo receptor, which should contribute to efficient N3.35 driven biased signaling. The M3.36 pattern in amniota AT2 favours the outward orientation of N3.35 and the receptor promiscuity. Both mutations have physiological consequences for the regulation of the renin-angiotensin system.

Homology Modeling of Class A G-Protein-Coupled Receptors in the Age of the Structure Boom.

With 700 members, G protein-coupled receptors (GPCRs) of the rhodopsin family (class A) form the largest membrane receptor family in humans and are the target of about 30% of presently available pharmaceutical drugs. The recent boom in GPCR structures led to the structural resolution of 57 unique receptors in different states (39 receptors in inactive state only, 2 receptors in active state only and 16 receptors in different activation states). In spite of these tremendous advances, most computational studies on GPCRs, including molecular dynamics simulations, virtual screening and drug design, rely on GPCR models obtained by homology modeling. In this protocol, we detail the different steps of homology modeling with the MODELLER software, from template selection to model evaluation. The present structure boom provides closely related templates for most receptors. If, in these templates, some of the loops are not resolved, in most cases, the numerous available structures enable to find loop templates with similar length for equivalent loops. However, simultaneously, the large number of putative templates leads to model ambiguities that may require additional information based on multiple sequence alignments or molecular dynamics simulations to be resolved. Using the modeling of the human bradykinin receptor B1 as a case study, we show how several templates are managed by MODELLER, and how the choice of template(s) and of template fragments can improve the quality of the models. We also give examples of how additional information and tools help the user to resolve ambiguities in GPCR modeling.

Evolution of chemokine receptors is driven by mutations in the sodium binding site.

Chemokines and their receptors (members of the GPCR super-family) are involved in a wide variety of physiological processes and diseases; thus, understanding the specificity of the chemokine receptor family could help develop new receptor specific drugs. Here, we explore the evolutionary mechanisms that led to the emergence of the chemokine receptors. Based on GPCR hierarchical classification, we analyzed nested GPCR sets with an eigen decomposition approach of the sequence covariation matrix and determined three key residues whose mutation was crucial for the emergence of the chemokine receptors and their subsequent divergence into homeostatic and inflammatory receptors. These residues are part of the allosteric sodium binding site. Their structural and functional roles were investigated by molecular dynamics simulations of CXCR4 and CCR5 as prototypes of homeostatic and inflammatory chemokine receptors, respectively. This study indicates that the three mutations crucial for the evolution of the chemokine receptors dramatically altered the sodium binding mode. In CXCR4, the sodium ion is tightly bound by four protein atoms and one water molecule. In CCR5, the sodium ion is mobile within the binding pocket and moves between different sites involving from one to three protein atoms and two to five water molecules. Analysis of chemokine receptor evolution reveals that a highly constrained sodium binding site characterized most ancient receptors, and that the constraints were subsequently loosened during the divergence of this receptor family. We discuss the implications of these findings for the evolution of the chemokine receptor functions and mechanisms of action.

Breast cancer in Tunisia: association of body mass index with histopathological aspects of tumors.

BACKGROUND: Previous studies have suggested a link between obesity and breast cancer (BC). However, there is no universal consensus, especially in population based studies. Because only few studies have been conducted on African women, we aimed here to assess the relationship between BMI at time of diagnosis and the BC histopathological features among Tunisian patients according to menopausal status using a hospital-based prospective cohort study. MATERIALS AND METHODS: Clinical and pathological data were collected from 262 patients stratified on four groups according to their BMI. The relationship between BMI and histopathological features at diagnosis was analysed using univariate and multivariate analysis. Receiver-operating characteristic (ROC) curves were used to evaluate the performance of BMI in predicting of high tumor grade, in comparison to ki-67 index of proliferation. RESULTS: Obesity was correlated with larger tumors, advanced grade and with ER-PR- Her2+ BC subtype. An association of BMI with tumor size and tumor grade was observed in both premenopausal and postmenopausal women. Additionally, a significant association between BMI and ER+, ER+PR+Her2+ and ER-PR-Her2+ status was revealed for premenopausal patients, while only ER+PR+Her2+ was associated with BMI for postmenopausal women. Finally, our results showed that compared to Ki67 proliferation index, BMI is a useful prognostic marker of high grade BC tumors. CONCLUSIONS: These data are the first to show that in Tunisia obese women suffering from BC have significantly larger tumors and advanced tumor grade and that higher BMI might influence tumor characteristics and behavior.

TR alpha 2 exerts dominant negative effects on hypothalamic Trh transcription in vivo.

Mammalian thyroid hormone receptors (TRs) have multiple isoforms, including the bona fide receptors that bind T3 (TRα1, TRß1 and TRß2) and a non-hormone-binding variant, TRα2. Intriguingly, TRα2 is strongly expressed in the brain, where its mRNA levels exceed those of functional TRs. Ablation of TRα2 in mice results in over-expression of TRα1, and a complex phenotype with low levels of free T3 and T4, without elevated TSH levels, suggesting an alteration in the negative feedback at the hypothalamic-pituitary level. As the hypothesis of a potential TRH response defect has never been tested, we explored the functional role of TRα2 in negative feedback on transcription of hypothalamic thyrotropin, Trh. The in vivo transcriptional effects of TRα2 on hypothalamic Trh were analysed using an in vivo reporter gene approach. Effects on Trh-luc expression were examined to that of two, T3 positively regulated genes used as controls. Applying in vivo gene transfer showed that TRα2 over-expression in the mouse hypothαlamus abrogates T3-dependent repression of Trh and T3 activation of positively regulated promoters, blocking their physiological regulation. Surprisingly, loss of function studies carried out by introducing a shTRα2 construct in the hypothalamus also blocked physiological T3 dependent regulation. Thus, modulating hypothalamic TRα2 expression by either gain or loss of function abrogated T3 dependent regulation of Trh transcription, producing constant transcriptional levels insensitive to feedback. This loss of physiological regulation was reflected at the level of the endogenous Trh gene, were gain or loss of function held mRNA levels constant. These results reveal the as yet undescribed dominant negative role of TRα2 over TRα1 effect on hypothalamic Trh transcription.

Relationship of thyroid function with obesity and type 2 diabetes in euthyroid Tunisian subjects.

OBJECTIVES: Although a relationship between obesity and metabolic consequences with thyroid function has been reported, the underlying pathogenesis is not completely known. In the current study, we evaluated the thyroid function in obese and/or diabetic patients compared to healthy normal weight peers, exploring the possible association between components of metabolic syndrome and thyroid function parameters. METHODS: We recruited 108 subjects (56 male and 52 female). In all subjects, thyroid stimulating hormone (TSH), free thyroxine (FT4), fasting plasma levels of insulin and glucose, homeostasis model assessment for insulin resistance, and obesity parameters were assessed. RESULTS: We found that circulating levels of TSH and FT4 were significantly increased in overweight and obese subjects. However, the data do not reveal any change of these hormones in diabetics. Multivariate linear regression analysis showed that TSH was directly associated with both obesity and insulin resistance parameters (p < 0.05). FT4 was negatively associated only with obesity parameters (p < 0.05). CONCLUSIONS: Our data strongly support that the changes of thyroid hormones may be influenced by adiposity and its metabolic consequences, such as insulin resistance. This relationship can be explained by a cross talk between adipose tissue release and thyroid function. Nevertheless, metformin treatment seems to affect thyroid function in diabetic patients by maintaining plasma thyrotropin levels to subnormal levels.

Human subcutaneous adipose tissue Glut 4 mRNA expression in obesity and type 2 diabetes.

Cellular resistance to insulin caused by reduced glucose transport and metabolism is a primary defect leading to the development of metabolic disease. While the etiology of insulin resistance is multifactorial, reduced insulin action is associated with impaired activity of the glucose transporter GLUT4 in insulin-sensitive tissues. Yet, the role of adipose tissue GLUT4 deregulation in the pathogenesis of insulin resistance, obesity, and diabetes is still unclear. In this study, we assessed the relative GLUT4 level in human subcutaneous adipose tissue from obese, diabetic, and diabetic obese versus control subjects, using a real-time PCR method. GLUT4 mRNA levels were considerably decreased among type 2 diabetic patients compared with those of the controls (P < 0.01), whereas no such difference was found between obese and normal-weight controls. Multiple linear regressions analysis in both diabetic non-obese and diabetic obese groups showed a negative correlation between GLUT4 mRNA expression and both markers of obesity or insulin resistance (P < 0.01). However, in obese group, GLUT4 was inversely associated only with HOMA-IR (P < 0.01). Our findings showed that adipose GLUT4 gene expression changes were more related to insulin resistance and type 2 diabetes rather than to obesity.

Relationship between subcutaneous adipose tissue expression of leptin and obesity in Tunisian patients.

BACKGROUND: The incidence of obesity has dramatically increased in overall the world. It is a consequence of imbalance between energy intake and energy expenditure. Leptin is a fat derived adipokine that has emerged over the past decade as a key hormone in the regulation of food intake and energy expenditure. Elevated leptin levels are found in obese humans, suggesting a role of leptin in regulating body weight and adiposity. AIM: The aim of this study was to investigate the change of leptin mRNA expression level and its correlation with obesity and several metabolic variables in Tunisian patients. METHODS: Real time quantitative polymerase chain reaction (QPCR) analysis was carried out among two groups who underwent an abdominal surgery: controls (n = 9) and obese patients (n = 7). RESULTS: Leptin mRNA expression in subcutaneous adipose tissue was markedly increased in obese patients (p < 0.01). It was positively correlated with measures of obesity waist circumference (WC) (r = 0, 71, p < 0.01) and body mass index (BMI) (r = 0, 68, p < 0.01). Interestingly, leptin gene expression was also correlated to insulin resistance index (r = 0, 72, p < 0.01). CONCLUSION: The present study is the first investigation of leptin regulation in subcutaneous adipose tissue of Tunisian population. Our data showed that leptin levels are higher in obese subjects than in control subjects. This indicates that the subcutaneous adipose plays an important role in impaired adipokine regulation, and consequently in developing metabolic disorder.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) modulates hypothalamic Trh regulation in vivo.

Thyroid hormone receptor (TR) and peroxisome proliferator-activated receptor gamma (PPARgamma) co-regulate numerous peripheral metabolic responses. To examine potential crosstalk between PPARgamma and TRbeta in the hypothalamus, thyrotropin-releasing hormone (Trh) regulation in the newborn mouse hypothalamus was followed. QPCR showed PPARgamma to be expressed in the hypothalamus at this developmental stage. Intracerebral injection of PPARgamma agonists modified transcription from a TRH-luc construct introduced into the hypothalamus and increased serum thyroxine levels. Furthermore, shRNA-based in vivo PPARgamma knockdown amplified T(3)-independent transcription and PPARgamma overexpression dose-dependently abrogated T(3)-dependent Trh repression. Overexpression of retinoid X receptor-alpha (RXRalpha), the common heterodimeric partner of PPARgamma and TRbeta, rescued PPARgamma abrogation of T(3)-dependent repression. Thus, competition for RXR could represent one mechanism underlying this hypothalamic crosstalk between PPARgamma and TRbeta. These demonstrations of PPARgamma effects on hypothalamic Trh transcription in vivo consolidate the role of the TRH neuron as a central integrator of energy homeostasis.

In vivo siRNA delivery to the mouse hypothalamus confirms distinct roles of TR beta isoforms in regulating TRH transcription.

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for evaluating gene function in vivo. In particular it should be able to provide tissue-specific and developmental stage-specific knock-down of target genes in physiological contexts. However, demonstrations of its use on neuronal specific genes in vivo are lacking. We examined whether a recently developed cationic lipid based approach was applicable to study the differential effects of the two beta thyroid hormone receptor (TR) isoforms, TRbeta1 and TRbeta2, on T3-transcriptional repression of the hypothalamic gene, TRH. The cationic lipid based technique used, JetSI/DOPE, was previously shown to efficiently knock-down reporter gene mRNA in vivo. Here we now show that its use to vectorise siRNA against TRbeta1 and TRbeta2 mRNA abrogates T3-mediated repression of hypothalamic TRH transcription. In particular, when using siRNA against either TRbeta1 or TRbeta2 differential effects are revealed. siRNA directed against TRbeta1 blocks both T3 independent activation and T3 dependent modulation of TRH transcription. In contrast, siRNA directed against TRbeta2 abrogates only T3 repression of transcription. These results corroborate our previous findings obtained in mutant TRbeta(-/-) mice, showing that the TRbeta1 and TRbeta2 isoforms have differential effects on T3-TRH transcription. The data thus show that the cationic lipid-based siRNA strategy can effectively be used to reveal fine, tissue specific and isoform specific effects on neuronal gene transcription in vivo.

[Transcriptional repression of the TRH gene]. / Répression transcriptionnelle du gène TRH.

The synthesis and secretion of thyroid hormones (TH: T3, T4) must be strictly regulated. TH act on their own production via a negative feedback system. The synthesis of thyrotropin-releasing hormone (TRH), produced in the hypothalamus, and thyrotropin (TSH) in the pituitary is inhibited at the transcriptional level by TH. TRH and TSH stimulate production of TH. An outstanding, still open, question is the molecular basis of T3-dependent transcription repression of TRH and TSH genes. However, some regulatory components have been identified, with the b-TH receptor (TRb) playing a specific regulatory role (versus TRa) in the negative feedback effects of T3 on production of TRH and TSH. Moreover, the N-terminus of TRb is known to be a key element in this regulation. A hypothesis to explain this isoform specificity could be that TRb and TRa interact differentially with transcriptional comodulators. Thus, it is critical to characterize these comodulators and to analyse their contribution to the transcription regulation of TRH.

Both thyroid hormone receptor (TR)beta 1 and TR beta 2 isoforms contribute to the regulation of hypothalamic thyrotropin-releasing hormone.

Thyroid hormones (TH) are essential regulators of vertebrate development and metabolism. Central mechanisms governing their production have evolved, with the beta-TH receptor (TRbeta) playing a key regulatory role in the negative feedback effects of circulating TH levels on production of hypothalamic TRH and hypophyseal TSH. Both TRbeta-isoforms (TRbeta1 and TRbeta2) are expressed in the hypothalamus and pituitary. However, their respective roles in TH-dependent transcriptional regulation of TRH are undefined. We confirmed the preferential role of TRbeta vs. TRalpha isoforms in TRH regulation in wild-type mice in vivo by using the TRbeta preferential agonist GC-1. We next determined the effects of tissue-specific rescue of TRbeta1 and TRbeta2 isoforms by somatic gene transfer in hypothalami of TRbeta null (TRbeta(-/-)) mice. TH-dependent TRH transcriptional repression was impaired in TRbeta(-/-) mice, but was restored by cotransfection of either TRbeta1 or TRbeta2 into the hypothalamus. TRbeta1, but not TRbeta2, displayed a role in ligand-independent activation. In situ hybridization was used to examine endogenous TRH expression in the paraventricular nucleus of the hypothalamus of TRbeta(-/-) or TRalpha null (TRalpha(o/o)) mice under different thyroid states. In contrast to published data on TRbeta2(-/-) mice, we found that both ligand-independent TRH activation and ligand-dependent TRH repression were severely impaired in TRbeta(-/-) mice. This study thus provides functional in vivo data showing that both TRbeta1 and TRbeta2 isoforms have specific roles in regulating TRH transcription.

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.