昆虫蜕皮激素受体研究进展

昆虫的蜕皮激素(molting hormone,MH)是甾醇类激素,在昆虫体内的活性形式为20-羟基蜕皮酮(20-hydroxyecdysone,20E)。在昆虫的正常发育过程中,昆虫的蜕皮、变态和繁殖受到蜕皮激素的调控。昆虫蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle,USP)均属于核受体超家族成员,具有核受体的结构特征,包括A/B域(转录激活域transactivation domain)、C域(DNA结合域DNA-binding domain,DBD)、D域(铰链域hinge region)、E域(配体结合域ligand binding domain,LBD)和F域。蜕皮激素受体在昆虫蜕皮、变态和繁殖等重要的生命过程中的级联反应启动位置,对昆虫的生长发育和繁殖的正常完成有着非常重要的作用。蜕皮激素通过与蜕皮激素受体和超气门蛋白组成的复合体相互作用,然后启动一系列级联反应的过程。本文介绍了EcR和USP的结构和功能,以及它们与蜕皮激素相互作用的机理,并对EcR在农业害虫防治等方面的应用进行了介绍,并讨论了研究中遇到的问题以及对未来的研究进行展望。     

蜕皮激素与其受体EcR-USP的转录调控机制

蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone, 20E)是一种典型的类固醇激素, 主导调控昆虫的蜕皮、变态、生殖等重要生理过程。20E受体EcR-USP已被鉴定近20年, 20E与其受体复合物的转录调控机制也有了许多重要突破。已有研究表明： （1）20E受体由核受体EcR和USP形成; （2）EcR-USP异源二聚体在分子伴侣蛋白复合物的协助下获得DNA结合活性; （3）20E通过解除共阻遏因子和募集共激活因子来激活EcR USP异源二聚体并启动下游基因的转录; (4）20E-EcR-USP配体-受体复合物引发20E初级应答基因的表达, 由20E初级反应基因编码的转录因子诱导表达的20E次级应答基因级联放大20E信号, 从而调控昆虫蜕皮、变态、生殖等生理过程。     

昆虫蜕皮激素信号转导途径研究进展

蜕皮与变态是全变态昆虫典型的发育特征。调控昆虫蜕皮与变态的激素主要有蜕皮激素和保幼激素。目前已经阐明了蜕皮激素的核受体EcR及部分核信号转导途径,但蜕皮激素是否存在膜受体及膜信号转导途径研究很少。研究证明,蜕皮激素存在细胞质中的信号转导分子和途径,蜕皮激素通过NTF2和Ran调控EcR入核启动基因转录。蜕皮激素使细胞质中的热休克蛋白Hsc70部分入核与USP结合启动基因转录。蜕皮激素通过蛋白激酶PKC使伴侣蛋白calponin磷酸化,参与蜕皮激素信号途径的基因转录。这些研究结果说明蜕皮激素除了有核受体和核受体信号转导途径外,还存在细胞膜受体和细胞膜信号转导途径。     

尺蠖蜕皮激素受体基因Eo-EcR 的克隆、生物信息学分析和表达检测

【目的】蜕皮激素受体（ecdysteroid receptor, EcR）是一种超家族核受体,它能与超气门蛋白USP组成异源二聚体复合物EcR/USP,调节20 羟基蜕皮酮（20E）的生物活性,进而调控昆虫的发育、变态及繁殖等生命过程。本研究旨在克隆茶尺蠖 Ectropis obliqua Prout EcR基因全长,并了解该基因的编码蛋白特征和时空表达模式。【方法】通过RT-PCR方法并结合RACE技术,克隆茶尺蠖EcR的基因全长,通过生物信息学软件和在线网站分析茶尺蠖EcR的生物学特性,通过实时荧光定量PCR（real-time quantitative PCR, qRT-PCR）技术比较茶尺蠖 EcR 在不同发育时期和6龄幼虫不同组织中的相对表达含量。【结果】克隆并鉴定了茶尺蠖EcR基因,将其命名为 Eo-EcR（基因登录号: KP869130.1）,Eo-EcR全长2 268 bp,含有1 728 bp开放阅读框,编码576个氨基酸。系统进化树和氨基酸同源性比对表明,Eo-EcR具有相对保守的进化特性,特别是与鳞翅目昆虫的保守性最高。三级结构模拟和功能结构域预测表明,Eo-EcR具有3个经典的结构模型,并以α螺旋为主,功能位点单一且为C₄型锌指结构。qRT-PCR结果表明,Eo-EcR在5龄和6龄幼虫期以及成虫期表达量较高,在其他龄期表达量变化不大;同时在前胸腺表达量最高,在血淋巴表达量最低。【结论】明确了Eo-EcR的核苷酸序列及编码蛋白特征,明确了Eo-EcR的时空表达特性。该研究结果为进一步研究Eo-EcR的分子功能和基于Eo-EcR为靶标杀虫剂的研制奠定分子基础。     

斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

【目的】蜕皮激素在昆虫变态发育中起着关键的调控作用。蜕皮激素活化为20-羟基蜕皮酮(20-hydroxyecdysone,20E)后与蜕皮激素受体二聚体〔蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle protein,USP)〕结合,启动20E诱导的级联反应。本研究旨在从互作蛋白角度探究EcR/USP自身调控的分子机理。【方法】以重要农业害虫斜纹夜蛾Spodoptera litura为研究对象,通过构建酵母双杂交筛选系统分别筛选了与EcR和USP相互作用的蛋白。【结果】文库转化效率达到3.0×106cfu/μg,文库滴度达到1.3×108cfu/m L,文库插入片段大小在500~2 000 bp之间。4种诱饵质粒(EcRA/p GBKT7,EcRB1/p GBKT7,USP1/p GBKT7和USP2/p GBKT7)对酵母细胞无毒性,并且无自激活性,说明构建的酵母双杂交文库质量可靠。用以上4种诱饵质粒筛选酵母双杂交文库,共得到110个互作蛋白,其中与EcRB1,USP1和USP2互作的蛋白分别有26,52和32个,未筛选到与EcRA互作的蛋白。随后,从中挑选了Dna J-5(Hsp40 homolog 5,一种热激蛋白分子伴侣),MBF2(mediator of Bm FTZ-F1 type 2,一种转录共激活子),polyubiquitin(多聚泛素类蛋白),esr16(ecdysteroid regulated 16k Da protein,一种蜕皮激素调控蛋白)和NEDD8-like(neural precursor cell expressed,developmentally down-regulated protein 8,一种泛素调节相关蛋白)5种蛋白,利用酵母双杂交和Far-Western印迹法进一步验证了蛋白间的互作关系。【结论】分子伴侣和泛素化修饰等在蜕皮激素受体调控中可能起着重要作用。本研究对深入理解昆虫变态发育的分子机理具有重要意义。     

斜纹夜蛾蜕皮激素受体与超气门蛋白的原核表达及活性检测

双酰基肼类杀虫剂模拟天然蜕皮激素作用影响幼虫蜕皮。昆虫蜕皮激素受体的高度敏感性和专一性要求必须建立新的杀虫活性检测技术, 以适应快速准确和大批量筛选的要求。本研究采用RT-PCR技术, 获取斜纹夜蛾Spodoptera litura蜕皮激素受体（EcR）与超气门蛋白（USP）功能域目的基因, 构建EcR、 USP功能区基因原核表达载体（pEHISEGFPTEV-EcR_cde和pEHISEGFPTEV-USP_cde）。载体经诱导表达和蛋白纯化, 获得EcR和USP功能区纯化蛋白。在蛋白浓度l mg/mL, ³H-PonA终浓度8 nmol/L的条件下, 采用放射性配基受体结合分析测定了4种药剂（虫酰肼、 呋喃虫酰肼、 抑食肼和灭幼脲）不同浓度下的放射性比活的变化。结果显示： 随着药剂浓度的逐渐增大, 前3种药剂的放射性比活都有不同程度的降低, 其中虫酰肼的放射性比活降低程度最大, 其次是呋喃虫酰肼和抑食肼, 灭幼脲的放射性比活基本无变化。这些结果表明相同条件下虫酰肼比呋喃虫酰肼和抑食肼有更高的杀虫活力, 本研究的方法可对双酰基肼类杀虫剂或者先导化合物进行初步筛选。     

保幼激素对昆虫变态发育调控的分子机制

近年来随着保幼激素(juvenile hormone,JH)核受体Methoprene tolerant(Met)被鉴定,JH对昆虫变态发育调控的分子机制的研究取得了极大的进展。本文在介绍Met的鉴定以及分子伴侣Hsp83和核孔蛋白Nup358对Met亚细胞定位调控的基础上,重点阐述了JH-Met-Kr-h1-Br信号通路在完全变态昆虫幼虫至蛹变态过程中的作用以及JH-Met-Kr-h1-E93信号通路在不完全变态昆虫和完全变态昆虫成虫羽化过程中的作用。此外,Met与蜕皮激素(20-hydroxyecdysone,20E)受体复合物EcR/USP的结合、Tai/SRC/FISC分别与Met和EcR/USP结合形成JH功能受体和20E功能受体复合物、JH对20E下游基因E75A的诱导以及USP与JH的结合等分子间的相互作用在JH与20E的互作中所产生的影响也将逐一进行论述。本文还对JH通过膜受体激活PKC和PLC等下游信号通路而发挥生理功能的研究进展进行了概述。     

昆虫成虫蜕皮激素研究进展

绝大多数成体昆虫羽化后,幼虫期间负责蜕皮激素合成的前胸腺即发生退化,但在一些内部生理及外部环境因子的调控下,某些成体组织(如生殖腺)可扮演类似前胸腺的角色合成与分泌蜕皮激素。蜕皮激素的功能发挥是经受体介导的,包括核受体(如EcR/USP)和膜受体(如DopEcR),它们广泛表达于成体许多组织,参与成虫行为、生殖、寿命、滞育及免疫应答等众多方面的调节,对维持基本的生理功能具有重要作用。就成虫蜕皮激素的产生组织及影响其滴度的因素、成虫蜕皮激素受体概述与组织分布、成虫蜕皮激素信号通路的功能发挥等研究进展方面加以综述。     

茶尺蠖蜕皮激素受体基因Eo-EcR的克隆、生物信息学分析和表达检测

【目的】蜕皮激素受体(ecdysteroid receptor,EcR)是一种超家族核受体,它能与超气门蛋白USP组成异源二聚体复合物EcR/USP,调节20-羟基蜕皮酮(20E)的生物活性,进而调控昆虫的发育、变态及繁殖等生命过程。本研究旨在克隆茶尺蠖Ectropis obliqua Prout EcR基因全长,并了解该基因的编码蛋白特征和时空表达模式。【方法】通过RT-PCR方法并结合RACE技术,克隆茶尺蠖EcR的基因全长,通过生物信息学软件和在线网站分析茶尺蠖EcR的生物学特性,通过实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)技术比较茶尺蠖EcR在不同发育时期和6龄幼虫不同组织中的相对表达含量。【结果】克隆并鉴定了茶尺蠖EcR基因,将其命名为Eo-EcR(基因登录号:KP869130.1),Eo-EcR全长2 268 bp,含有1 728 bp开放阅读框,编码576个氨基酸。系统进化树和氨基酸同源性比对表明,Eo-EcR具有相对保守的进化特性,特别是与鳞翅目昆虫的保守性最高。三级结构模拟和功能结构域预测表明,Eo-EcR具有3个经典的结构模型,并以α螺旋为主,功能位点单一且为C4型锌指结构。qRT-PCR结果表明,Eo-EcR在5龄和6龄幼虫期以及成虫期表达量较高,在其他龄期表达量变化不大;同时在前胸腺表达量最高,在血淋巴表达量最低。【结论】明确了Eo-EcR的核苷酸序列及编码蛋白特征,明确了Eo-EcR的时空表达特性。该研究结果为进一步研究Eo-EcR的分子功能和基于Eo-EcR为靶标杀虫剂的研制奠定分子基础。     

蜕皮激素和IIS-TORC1信号的分子互作

蜕皮激素信号主导调控昆虫的蜕皮和变态,决定昆虫的发育时间;IIS-TORC1信号整合生长因子、激素、营养和能量信号,决定昆虫的生长速率。蜕皮激素和IIS-TORC1信号之间发生3种分子互作:(1)IIS-TORC1信号促进前胸腺和卵巢合成蜕皮激素前体。(2)在蜕皮和变态期间,蜕皮激素抑制脂肪体细胞内IIS-TORC1信号、Myc的转录、细胞生长及其内分泌功能,导致脑神经分泌细胞分泌胰岛素样肽的功能减弱,从而降低昆虫全身性的IIS-TORC1信号。(3)在幼虫摄食期间,胰岛素信号抑制FOXO的转录活性,降低了蜕皮激素受体EcR的转录共激活因子DOR编码基因的转录水平,从而阻碍了蜕皮激素信号传导。蜕皮激素信号和IIS-TORC1信号协同调控发育时间和生长速率共同决定昆虫的个体大小。     

Highly flexible ligand binding pocket of ecdysone receptor: a single amino acid change leads to discrimination between two groups of nonsteroidal ecdysone agonists

The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.     

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

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.     

Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).     

Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.