The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule

Dual FRET assay for detecting receptor protein interaction with DNA

We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein–protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27pal response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5′ half-site in the presence of EcRDBD when the 3′ half-site was occupied, and increased affinity of EcRDBD to the 3′ half-site when the 5′ half-site was occupied

The role of retinoid X receptors in the control of emotional processes in mice

Rxry est l un des récepteurs nucléaires impliqués dans la signalisation à l acide rétinoïque. L ablation de Rxry chez les souris provoque le développement de comportements de type dépressifs - désespoir et d anhédonie. De tels déficits pouvaient être normalisés par des anti-dépresseurs tels que la fluoxetine, suggérant donc l importance de telles données pour la recherche sur la dépression.Nous avons trouvé que le NAcSh est une structure impliquée dans le contrôle par Rxry des comportements motivés étant donné que la ré-expression de Rxry dans cette structure par le virus normalise les déficits comportementaux chez les souris Rxry-/-. Nous avons démontré que le récepteur Drd2 qui est sous-exprimé dans le NAcSh des souris Rxry-/- est nécessaire dans le contrôle des comportements affectifs étant donné que le blocage des activités du Drd2 par infusion de raclopride dans le NAcSh empêche le rétablissement du phénotype Rxry-/- par le virus AAV2-Rxry Cette observation est étayée par le rétablissement fonctionnel des déficits comportementaux par injection de virus ou traitement à la fluoxetine qui augmentent l expression du Drd2 dans le NAcSh chez les souris Rxry-/-. Ces données sont la première démonstration que les récepteurs aux rétinoides sont impliqués dans le contrôle des comportements affectifs chez la souris.Nous avons observé que l ablation de Rxry provoquent une hyperactivité du NAcSh. Nous avons observé des phénomènes similaires dans un modèle de stresse par défaite sociale. L existence de telle corrélation dans deux modèles animaux distincts de comportements dépressifs suggère que l hyperactivité du NacSh pourrait être un phénomène commun sous-tendant la dépression.Rxry is one of nuclear receptors involved in retinoic acid signalling. Ablation of this receptor in mice leads to development of depressive-like behaviours - despair and anhedonia. Importantly, such deficits could be normalized by antidepressant, fluoxetine chronic treatment, suggesting thus the relevance of such data for research into depression. We identified that NAcSh is a key structure implicated in Rxry control of motivated behaviours as virus mediated re-expression of Rxry in this brain region normalized behavioural deficits of Rxry-/- mice. We demonstrated that dopaminergic D2 receptor Drd2, which is underexpressed in the NAcSh of Rxry-/- mice is necessary for Rxry control of affective behaviours since blocking of Drd2 activities by infusion of raclopride into the NAcSh prevented AAV2-Rxry mediated rescue of Rxry-/- phenotype. This observation was further supported by functional rescue of behavioral deficits by virus mediated or chronic fluoxetine treatment increase of Drd2 expression in the NAcSh of Rxry-/- mice. Such data provide the first evidence that retinoid receptors are implicated in the control of affective behaviours in mice.We also identified that molecular changes caused by Rxry ablation lead to hyperactivity of the NAcSh. Importantly, we observed similar phenomenon in etiologically different model of depression social defeat stress model. Existence of such correlation in two distinct animal models of depressive behaviours, suggests that NAcSh hyperactivity might be a common phenomenon underlying depression.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Molecular mechanism of calcium induced trimerization of C1q-like domain of otolin-1 from human and zebrafish

Abstract The C1q superfamily includes proteins involved in innate immunity, insulin sensitivity, biomineralization and more. Among these proteins is otolin-1, which is a collagen-like protein that forms a scaffold for the biomineralization of inner ear stones in vertebrates. The globular C1q-like domain (gC1q), which is the most conserved part of otolin-1, binds Ca2+ and stabilizes its collagen-like triple helix. The molecular details of the assembly of gC1q otolin-1 trimers are not known. Here, we substituted putative Ca2+-binding acidic residues of gC1q otolin-1 with alanine to analyse how alanine influences the formation of gC1q trimers. We used human and zebrafish gC1q otolin-1 to assess how evolutionary changes affected the function of the protein. Surprisingly, the mutated forms of gC1q otolin-1 trimerized even in the absence of Ca2+, although they were less stable than native proteins saturated with Ca2+. We also found that the zebrafish gC1q domain was less stable than the human homologue under all tested conditions and became stabilized at higher concentrations of Ca2+, which showed that specific interactions leading to the neutralization of the negative charge at the axis of a gC1q trimer by Ca2+ are required for the trimers to form. Moreover, human gC1q otolin-1 seems to be optimized to function at lower concentrations of Ca2+, which is consistent with reported Ca2+ concentrations in the endolymphs of fish and mammals. Our results allow us to explain the molecular mechanism of assembly of proteins from the C1q superfamily, the modulating role of Ca2+ and expand the knowledge of biomineralization of vertebrate inner ear stones: otoliths and otoconia