Structure-based analysis of the ultraspiracle protein and docking studies of putative ligands

The ultraspiracle protein (USP) is the insect ortholog of the mammalian retinoid X receptor (RXR). Fundamental questions concern the functional role of USP as the heterodimerization partner of insect nuclear receptors such as the ecdysone receptor. The crystallographic structures of the ligand binding domain of USPs of Heliothis virescens and Drosophila melanogaster solved recently show that helix 12 is locked in an antagonist conformation raising the question whether USPs could adopt an agonist conformation as observed in RXRα. In order to investigate this hypothesis, a homology model for USP is proposed that allows a structural analysis of the agonist conformation of helix 12 based on the sequence comparison with RXR. For USP, one of the main issues concerns its function and in particular whether its activity is ligand independent or not. The x-ray structures strongly suggest that USP can bind ligands. Putative ligands have therefore been docked in the USP homology model. Juvenile hormones and juvenile hormone analogs were chosen as target ligands for the docking study. The interaction between the ligand and the receptor are examined in terms of the pocket shape as well as in terms of the chemical nature of the residues lining the ligand binding cavity

Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue

International audienceLow-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto-and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes. Citation: Nativel B, Marimoutou M, Thon-Hon VG, Gunasekaran MK, Andries J, et al. (2013) Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue. PLoS ONE 8(9): e76039

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

NMR structures of the glutaredoxin (GLXR) domains from Br. melitensis and Ba. henselae have been determined as part of the SSGCID initiative. Comparison of the domains with known structures reveals overall structural similarity between these proteins and previously determined E. coli GLXR structures, with minor changes associated with the position of helix 1 and with regions that diverge from similar structures found in the closest related human homolog

Molecular Evolution of Ultraspiracle Protein (USP/RXR) in Insects

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (dN/dS), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that dS is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand

Negative Regulation of Endogenous Stem Cells in Sensory Neuroepithelia: Implications for Neurotherapeutics

Stem cell therapies to treat central nervous system (CNS) injuries and diseases face many obstacles, one of which is the fact that the adult CNS often presents an environment hostile to the development and differentiation of neural stem and progenitor cells. Close examination of two regions of the nervous system – the olfactory epithelium (OE), which regenerates, and the neural retina, which does not – have helped identify endogenous signals, made by differentiated neurons, which act to inhibit neurogenesis by stem/progenitor cells within these tissues. In this chapter, we provide background information on these systems and their neurogenic signaling systems, with the goal of providing insight into how manipulation of endogenous signaling molecules may enhance the efficacy of stem cell neurotherapeutics

Etude de la relation structure et fonction du récepteur à l'ecdysone

Les récepteurs nucléaires d'hormones sont des facteurs de transcription dont l'activité est ligand-dépendante. Le récepteur fonctionnel à l'ecdysone des insectes est constitué d'un dimère de récepteurs nucléaires associant EcR (Ecdysone Receptor) à son partenaire USP (Ultraspiracle), homologue de RXR, le récepteur à l'acide rétinoïque9-cis des cordés. Le domaine de fixation du ligand (LBD) de USP présente une divergence de séquence majeure chez un groupe d'insectes " supérieurs " comprenant les diptères (mouches, moustiques) et les lépidoptères (papillons).Au cours de ma thèse, j'ai contribué à la validation fonctionnelle par mutagenèse ponctuelle et transactivation en cellules eucaryotes de la première structure du récepteur à l'ecdysone (H. virescens, lépidoptère) qui a révélé l'existence chez ECR de deux poches de fixation du ligand se recoupant partiellement.D'autre part, afin de comprendre l'évolution particulière du LBD de USP chez les insectes, j'ai initié une étude comparative des rapports structure-fonction de USP de Tribolium castaneum. En effet, USP de Tribolium(coléoptère) est représentatif de celui des insectes non diptères/lépidoptères : sa séquence peptidique est beaucoup plus proche de celle du RXR humain que de celle d'un USP de diptères ou de lépidoptères. En comparant les propriétés de USP de Tribolium et RXR humain par transactivation et spectrométrie de masse, j'ai pu montrer que USP de Tribolium n'est pas activé par l'acide rétinoïque 9-cis mais fixe des acides gras.La résolution de la structure cristalline du LBD de TcUSP à 2,71 Å révèle une poche dépourvue de ligand fort différente des conformations apo déjà observées chez des RXR de cordés. Un résidu conservé de manière différentielle semble être à l'origine de la discrimination des ligands par USP.Nuclear receptors are transcription factors with a ligand-dependant activity. The Insect functional ecdysone receptor is a nuclear receptors heterodimer constituted by EcR (Ecdysone Receptor) and its partner USP(Ultraspiracle), which is the homologue of RXR, the 9-cis Retinoic Acid receptor of chordates. The ligand Binding Domain (LBD) of USP presents a major sequence divergence in a group of "higher insects" comprising dipterans (flies, mosquitoes) and lepidopterans (moths, butterflies).The first structure of the ecdysone receptor has been solved for H. virescens (Lepidoptera) in the laboratory andreveals for EcR two different and partially overlapping ligand binding pockets. During my PhD, I have performed a functional validation of this structure using punctual mutagenesis and transactivation tests in eukaryotic cells.After that, in order to understand the particular evolution of the USP LBD among insects, I have initiated acomparative structure-function study of Tribolium castaneum (coleoptera) USP. Actually, the Tribolium USP is representative of non dipteran/lepidopteran insects USP : its peptidic sequence is much closer to the one of human RXR than to Diptera and Lepidoptera USP.The properties comparison of Tribolium USP and human RXR using transactivation tests and mass spectrometry shows that Tribolium USP is not activated by 9-cis Retinoic Acid but does bind fatty acids.The crystal structure of TcUSP LBD at a 2,71 Å resolution reveals an empty ligand binding pocket. The conformation is quite different from the ones of previously solved chordates RXR apo structures. A differentially conserved residue seems to be responsible of the discrimination of ligands by USP.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Rapid divergence of the ecdysone receptor in Diptera and Lepidoptera suggests coevolution between ECR and USP-RXR.

Ecdysteroid hormones are major regulators in reproduction and development of insects, including larval molts and metamorphosis. The functional ecdysone receptor is a heterodimer of ECR (NR1H1) and USP-RXR (NR2B4), which is the orthologue of vertebrate retinoid X receptors (RXR alpha, beta, gamma). Both proteins belong to the superfamily of nuclear hormone receptors, ligand-dependent transcription factors that share two conserved domains: the DNA-binding domain (DBD) and the ligand-binding domain (LBD). In order to gain further insight into the evolution of metamorphosis and gene regulation by ecdysone in arthropods, we performed a phylogenetic analysis of both partners of the heterodimer ECR/USP-RXR. Overall, 38 USP-RXR and 19 ECR protein sequences, from 33 species, have been used for this analysis. Interestingly, sequence alignments and structural comparisons reveal high divergence rates, for both ECR and USP-RXR, specifically among Diptera and Lepidoptera. The most impressive differences affect the ligand-binding domain of USP-RXR. In addition, ECR sequences show variability in other domains, namely the DNA-binding and the carboxy-terminal F domains. Our data provide the first evidence that ECR and USP-RXR may have coevolved during holometabolous insect diversification, leading to a functional divergence of the ecdysone receptor. These results have general implications on fundamental aspects of insect development, evolution of nuclear receptors, and the design of specific insecticides

Molecular adaptation and resilience of the insect's nuclear receptor USP

[Background] The maintenance of biological systems requires plasticity and robustness. The function of the ecdysone receptor, a heterodimer composed of the nuclear receptors ECR (NR1H1) and USP (NR2B4), was maintained in insects despite a dramatic divergence that occurred during the emergence of Mecopterida. This receptor is therefore a good model to study the evolution of plasticity. We tested the hypothesis that selection has shaped the Ligand-Binding Domain (LBD) of USP during evolution of Mecopterida.[Results] We isolated usp and cox1 in several species of Drosophilidae, Tenebrionidae and Blattaria and estimated non-synonymous/synonymous rate ratios using maximum-likelihood methods and codon-based substitution models. Although the usp sequences were mainly under negative selection, we detected relaxation at residues located on the surface of the LBD within Mecopterida families. Using branch-site models, we also detected changes in selective constraints along three successive branches of the Mecopterida evolution. Residues located at the bottom of the ligand-binding pocket (LBP) underwent strong positive selection during the emergence of Mecopterida. This change is correlated with the acquisition of a large LBP filled by phospholipids that probably allowed the stabilisation of the new Mecopterida structure. Later, when the two subgroups of Mecopterida (Amphiesmenoptera: Lepidoptera, Trichoptera; Antliophora: Diptera, Mecoptera, Siphonaptera) diverged, the same positions became under purifying selection. Similarly, several positions of the heterodimerisation interface experienced positive selection during the emergence of Mecopterida, rapidly followed by a phase of constrained evolution. An enlargement of the heterodimerisation surface is specific for Mecopterida and was associated with a reinforcement of the obligatory partnership between ECR and USP, at the expense of homodimerisation.[Conclusions] In order to explain the episodic mode of evolution of USP, we propose a model in which the molecular adaptation of this protein is seen as a process of resilience for the maintenance of the ecdysone receptor functionality.Support for this research was provided by CNRS, ENS de Lyon, ANR, the Spanish Ministry of Science and Innovation (projects CGL2008-03517/BOS to X.B., and BFU2006-13212 and BFU2009-10571 to D.M.) and by the CSIC (project 2010TW0019 to X.B.). O.M. is recipient of a pre-doctoral research grant from Generalitat de Catalunya.Peer Reviewe

Recombinant human HSP60 produced in ClearColi™ BL21(DE3) does not activate the NFκB pathway

International audienceHSP60, an intracellular molecular chaperone has been largely described as an alarmin or damage-associated molecular pattern when released outside the cell. HSP60 has been reported as a possible ligand of TLR2 or TLR4 inducing NFκB-dependant signaling pathway leading to cytokine secretion. However, recent publications suggested that HSP60 could not act as an activator of TLR4 by itself. The observed effect could be due to the presence of endotoxin in HSP60 preparation especially LPS. In order to clarify the controversy, we produced recombinant human HSP60 in two different strains of Escherichia coli, standard strain for protein overproduction, BL21(DE3), and the new ClearColi BL21(DE3) strain which lacks LPS-activity through TLR4. Undoubtedly, we have shown that recombinant HSP60 by itself was not able to induce NFκB-dependant signaling pathway in a model of THP1 monocyte cell line. Our data suggest that HSP60 needs either pathogen-associated molecules, specific post-translational modification and/or other host factors to activate immune cells via NFκB activation