Récepteur nucléaire | EPFL Graph Search

Les récepteurs nucléaires sont une super-famille de récepteurs biochimiques. Ce sont des protéines actives dans le noyau des cellules qui, pour nombre d'entre elles, transmettent à celles-ci des signaux hormonaux spécifiques conduisant à la modulation de l'expression de gènes cibles. Les récepteurs nucléaires sont présents chez tous les métazoaires. La famille est constituée de 49 membres identifiés à ce jour chez l'homme mais de plus d'une centaine toutes espèces confondues. Modes d'action des récepteurs nucléaires De nombreux récepteurs nucléaires fonctionnent avant tout comme des facteurs de transcription inductibles par un ligand (comme une hormone stéroïde, mais aussi par exemple certains lipides intracellulaires). Cependant beaucoup n'ont pas de ligand identifié, ils sont dits récepteurs nucléaires orphelins. De plus, certains récepteurs nucléaires ont des effets autrement que par leur qualité de facteurs de transcription. Ils peuvent par exemple agir sur d'autres v

In vivo investigation of estrogenic activity by fluorescence techniques

Hanna Jankevics

Hormone binding nuclear receptors are transcription factors that activate gene transcription by binding to specific DNA sequences in the gene promoter region and then recruiting the necessary coactivator proteins for transcriptional activation. The knowledge of the function of these receptors, e.g. how they activate or repress transcription are deduced mainly from in vitro biochemical experiments. A general model has been proposed that these receptors and the recruited coactivator proteins form relatively stable complexes with gene promoters. Only recently, in the last four years, additional in vivo data measured with fluorescence recovery after photobleaching (FRAP) have shown that many hormone receptors and coactivators are very mobile inside the nucleus and their association to chromatin is very dynamic, much faster than what has been anticipated from earlier biochemical studies. Although the mobility is reported to be high for the hormone binding receptors, there are consistent reports about reduced mobility in the presence of agonists and antagonists. However, little is known about the significance of the altered mobility. Open questions are, whether the change of mobility is due to increasing interactions with other proteins, e.g. by forming larger protein complexes or if it is due to interactions with chromatin and other relative immobile domains of the nucleus. In this work, fluorescence correlation spectroscopy (FCS) has been employed to monitor the mobility of both a hormone binding receptor, the estrogen receptor (ER), and coactivator proteins belonging to the steroid receptor coactivator (SRC)-family inside the nucleus, to investigate how their mobility is influenced by different ligands. The main advantages of FCS compared to FRAP are (i) its sensitivity which enables measurements of proteins of low abundance in live cells and (ii) its higher temporal resolution which allows us to resolve different diffusing species in the millisecond range. This work has been divided into four major parts. The first contains the construction and characterization of activity of fluorescently labeled molecules. The second part describes the optimization of the experimental conditions for measuring the mobility of yellow fluorescent protein (YFP) in living cells. The third part reports on the effects of YFP-labeled ER inside the nucleus in the presence of different ligands. Finally the fourth part investigates the effects of different ligands on the interaction between receptor and coactivator in living cells. The main results of this thesis are the following: The finding of the different mobility patterns of YFP-ER in the presence of agonists and antagonists indicate that the different ligands induce multiple discrete diffusive states; some are re-occuring for different ligands while others are characteristic for particular ligands. The potency of the different ligands to induce a lower mobility of the YFP-ER was estimated, and the relative order of the potencies to induce the lower mobility corresponds well to the relative order of their affinities for ER. ER and coactivator proteins were shown to interact in the absence of ligands and there are also clear indications of the co-existence of multiple ER-SRC complexes in the presence of agonists. The full antagonist abolishes these interactions completely in the case of the full-length coactivator and only partly when a truncated coactivator, containing only the receptor interaction domain, was used.

EPFL2004

Characterizing molecular recognition principles of specific odorant molecule interactions with an olfactory receptor and a nuclear hormone receptor

Sylvain Etter

Olfaction, the detection of odorous chemicals in the environment, is one of the oldest mammalian sensory systems and involves large number (up to 1000) of distinct G protein coupled olfactory receptors (OR). The chemical interaction of volatile molecules with specific ORs marks the primary step in odor perception and is of critical importance for elucidating the molecular basis of detecting and discriminating thousands of odorous chemicals. Despite the fact that a number of putative ORs have been cloned, only limited progress has been made in assigning odorant molecules to specific receptors. Furthermore, the analysis of OR–ligand interactions at a cellular level is complicated by combinatorial recognition principles and thus requires large scale odorant and receptor screening to establish receptor-specific ligand profiles. Described herein are efforts towards the development of reliable and efficient assays for detecting and quantifying the responses of heterologously expressed ORs to odorants. Three strategies were employed for the optical or electrophysiological detection of ORs responses with functional assays in HEK293 cells. In two approaches, OR activation was monitored via the endogenous cAMP pathway, which was coupled either to the cAMP response element-mediated expression of different reporter genes, or to a cyclic nucleotide gated ion channel. In a third approach, OR activation was measured using Ca2+ imaging in the presence of a promiscuous Gαq protein. ORs of different species were then functionally analyzed using the different assays. A library of 250 odorant compounds representative of the chemical functional groups relevant in perfumery was screened; 16 novel specific ligands for the mouse eugenol odorant receptor (EGOR) were identified. Ten of these compounds are structurally unrelated to previously identified ligands and thus provide an extended molecular basis for describing the ligand-specificity and selectivity of EGOR in new detail. Interestingly, some ORs exhibited odorant responses in the cAMP-based assays, but not in Ca2+ imaging assays and vice-versa, which indicates a possible preferential OR coupling to specific G proteins. Such preferential coupling might be dependent on the cell type or influenced by specific odorant molecules and could potentially increase the complexity in odorantmediated signaling. A second part of this thesis describes investigations into other potential activities of odorants apart from their role in olfaction. An odorant compound library was screened to determine possible xenoestrogenic activity on human ERα expressed in cultured mammalian cells. An image-based assay to observe the nuclear redistribution processes of a functional YFP-ERα fluorescent chimera allowed the identification of seven odorant molecules that interact with the receptor. As the optical detection of YFP-ERα redistribution processes did not distinguish between agonistic and antagonistic effects, we tested the active substances in a reporter assay using luciferase under transcription control of two estrogen response elements (ERE). Two active odorants identified in the screening exhibited agonist activity as indicated by a dose-dependent activation of ERα mediated transcription of the luciferase reporter gene.

EPFL2007

Ablation of the Tumor Suppressor Histidine Triad Nucleotide Binding Protein 1 is Protective Against Hepatic Ischemia/Reperfusion Injury

Gisèle Ferrand

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. Conclusion: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury. (HEPATOLOGY 2011;53:243-252)

2011