Influence of hormone on intracellular localization of the Drosophila melanogaster ecdysteroid receptor (EcR)

In the absence of hormone the ecdysteroid receptor (EcR) is distributed between the cytoplasm and the nucleus. Addition of the hormone muristerone A increases nuclear localization of wild type EcR within 5–10 min. Mutation of M504 to alanine, an amino acid, which is essential for ligand binding and which is situated in helix 5 of the ligand binding domain, abolishes hormone binding but still allows nuclear localization at only slightly reduced levels in the absence of hormone, whereas nuclear localization of EcR^M504R is nearly abolished. Cotransfection with ultraspiracle (USP), the invertebrate ortholog of RXR, leads to exclusively nuclear localization of wild type EcR and EcR^M504A indicating that basal heterodimerization in the absence of hormone is still possible. In the presence of Usp, EcR^M504R is only partially localized in the nucleus. EMSA experiments show that the ligand muristerone A enhances binding of wild type EcR, but only slighthly of mutated EcRs, to the canonical hsp 27 ecdysone response element. This is confirmed by transactivation studies. The results indicate that the architecture of the E-domain of EcR is important for nuclear localization even in the absence of a ligand.     

昆虫蜕皮激素受体及其类似物的杀虫机制研究进展

昆虫的蜕皮、变态和繁殖受到蜕皮激素的严格调控。蜕皮激素作用靶标由蜕皮激素受体（ecdysteroid receptor, EcR）和超气门蛋白（ultraspiracle protein, USP）组成,蜕皮激素与EcR/USP作用启动蜕皮级联反应过程。昆虫EcR具有种类或类群的特异性,研究其结构、功能和调控机理在开发环境友好型新药剂和基因调控开关等方面具有重要指导作用。该文介绍了昆虫EcR的结构和功能特点,蜕皮激素及其类似物与EcR/USP的分子作用方式,以及基于EcR/USP的新杀虫剂创制和基因调控开关设计等方面的重要进展。     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.     

Iodination of the progesterone receptor from hen oviduct spares the DNA-binding domain

The progesterone receptor from hen oviduct is isolated as a complex of two subunits, A and B. The A protein binds one molecule of progesterone and also binds to DNA with high affinity. The native A protein can be labeled with iodine with no loss of DNA binding activity. Limited Staphylococcus aureus V8 protease digestion of the labeled preparation results in a number of DNA-binding and non-DNA-binding fragments of the receptor. The progesterone-binding domain contains iodine label. However, two low-molecular-weight DNA-binding fragments do not contain iodine label, indicating a lack of susceptible tyrosine residues near the DNA-binding site of the native receptor. The labeled receptor and its fragments will facilitate studies of the isolated DNA-binding and progesterone-binding domains of the hen A protein as well as of the activity of the native receptor in the presence and absence of hormone.     

The ubiquitous nature of the progesterone receptor binding factor-1 (RBF-1) in avian tissues

The avian oviduct receptor binding factor-1 (RBF-1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF-1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF-1. The heart and pancreas had the highest and lowest RBF-1 levels, respectively; the concentration ranging by ～ 50-fold in these tissues. The 10 kDa size of the RBF-1 detected in all tissues suggests no significant tissue-specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF-1 have identical amino-terminal sequence. Using a recently isolated cDNA to RBF-1, the levels of RBF-1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue-specific differences in concentration. The presence of RBF-1 in non-progesterone responsive tissues suggests the possibility that RBF-1 may not be specifically involved in PR-DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor. © 1994 Wiley-Liss, Inc.     

雌激素受体亚型及其配体调节基因转录机制的研究

本文综述雌激素受体亚型（ERα和ERβ）的结构,功能,组织分布,生理作用及雌激素受体配体调节基因转录的机制,目的是深入系统地了解植物雌激素和选择性雌激素受体调节剂的作用路径及其组织特异性的发生机制,最终为提高雌激素类药物的选择性,优化以临床为基础的药物设计提供一条较为系统的思路。结果表明,ERα和ERβ对不同雌激素类化合物产生不同应答,配体的结构不同,调节基因转录的路径不同和募集的辅调节蛋白的不同是雌激素受体两种亚型组织特异性激活或抑制的主要原因。     

Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)

Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.     

麦红吸浆虫蜕皮激素受体（EcR）基因的克隆与表达分析

为了研究蜕皮激素受体（EcR）在麦红吸浆虫Sitodiplosis mosellana (Géhin)滞育活动中的作用, 利用RT PCR和RACE技术克隆得到了麦红吸浆虫蜕皮激素受体基因cDNA全长, 并通过Real-time quantitative PCR研究了其表达情况。该cDNA全长序列被命名为SmEcR （GenBank登录号： KC491135）, 其开放阅读框长1 386 bp, 编码461个氨基酸残基。其蛋白预测分子量52.90 kD, 理论等电点6.24。该蛋白与其他已报道的昆虫EcR蛋白具有很高的同源性, 其中与迟眼蕈蚊Bradysia coprophila中相应蛋白的氨基酸序列一致性高达92%。SmEcR在麦红吸浆虫不同滞育时期和不同虫态中均有表达, 且在不同滞育时期、 不同虫态中的表达量差异很大。在滞育不同时期以11月表达量最高, 12月表达量最低; 在不同虫态以麦穗幼虫中的表达量较低, 而成虫中的表达水平很高。本研究为进一步明确SmEcR在麦红吸浆虫滞育调控中的作用奠定了基础。     

Artificial miRNA-mediated silencing of ecdysone receptor (EcR) affects larval development and oogenesis in Helicoverpa armigera

The insect pests are real threat to farmers as they affect the crop yield to a great extent. The use of chemical pesticides for insect pest control has always been a matter of concern as they pollute the environment and are also harmful for human health. Bt (Bacillus thuringensis) technology helped the farmers to get rid of the insect pests, but experienced a major drawback due to the evolution of insects gaining resistance towards these toxins. Hence, alternative strategies are high on demand to control insect pests. RNA-based gene silencing is emerging as a potential tool to tackle with this problem. In this study, we have shown the use of artificial microRNA (amiRNA) to specifically target the ecdysone receptor (EcR) gene of Helicoverpa armigera (cotton bollworm), which attacks several important crops like cotton, tomato chickpea, pigeon pea, etc and causes huge yield losses. Insect let-7a precursor miRNA (pre-miRNA) backbone was used to replace the native miRNA with that of amiRNA. The precursor backbone carrying the 21 nucleotide amiRNA sequence targeting HaEcR was cloned in bacterial L4440 vector for in vitro insect feeding experiments. Larvae fed with Escherichia coli expressing amiRNA-HaEcR showed a reduction in the expression of target gene as well as genes involved in the ecdysone signaling pathway downstream to EcR and exhibited mortality and developmental defects. Stem-loop RT-PCR revealed the presence of amiRNA in the insect larvae after feeding bacteria expressing amiRNA-HaEcR, which was otherwise absent in controls. We also found a significant drop in the reproduction potential (oogenesis) of moths which emerged from treated larvae as compared to control. These results demonstrate the successful use of an insect pre-miRNA backbone to express amiRNA for gene silencing studies in insects. The method is cost effective and can be exploited as an efficient and alternative tool for insect pest management.     

Crystal structure of the vitamin D nuclear receptor ligand binding domain in complex with a locked side chain analog of calcitriol

The crystal structures of vitamin D nuclear receptor (VDR) have revealed that all compounds are anchored by the same residues to the ligand binding pocket (LBP). Based on this observation, a synthetic analog with a locked side chain (21-nor-calcitriol-20(22),23-diyne) has been synthesized in order to gain in entropy energy with a predefined active side chain conformation. The crystal structure of VDR LBD bound to this locked side chain analogue while confirming the docking provides a structural basis for the activity of this compound.