RESUMEN

Coleoptera is the largest taxa of animals by far. The robust reproductive capacity is one of the main reasons for such domination. Successful female reproduction partially relies on effective vitellogenesis. However, the hormone regulation of vitellogenesis remains to be explored. In the present paper, in vitro culture of Leptinotarsa decemlineata 1-day-old adult fat bodies in the 20E-contained median did not activate juvenile hormone production and insulin-like peptide pathways, but significantly stimulated the expression of two LdVg genes, in a cycloheximide-dependent pattern. In vivo RNA interference (RNAi) of either ecdysone receptor (LdEcR) or ultraspiracle (Ldusp) by injection of corresponding dsRNA into 1-day-old female adults inhibited oocyte development, dramatically repressed the transcription of LdVg genes in fat bodies and of LdVgR in ovaries; application of JH into the LdEcR or Ldusp RNAi L. decemlineata females did not restore the oocyte development, partially rescued the decreased LdVg mRNA levels but over-compensated LdVgR expression levels. The same RNAi experiments were performed in another Coleoptera species, Henosepilachna vigintioctopunctata. Little yolk substances were seen in the misshapen oocytes in the HvEcR or Hvusp RNAi ovaries, in contrast to larger amounts of yolk granules in the normal oocytes. Correspondingly, the transcript levels of HvVg in the fat bodies and ovaries decreased significantly in the HvEcR and Hvusp RNAi samples. Our results here show that 20E signaling is indispensable in the activation of vitellogenesis in the developing oocytes of the two beetle species.

RESUMEN

Diaphorina citri Kuwayama is of great concern because of its ability to transmit devastating citrus greening illness (Huanglongbing). One strategy for controlling HLB may involve limiting the spread of D. citri. Insecticides using dsRNA target genes may be a useful option to control D. citri. The ecdysone receptor (EcR) and ultraspiracle (USP) are crucial for the growth and reproduction of insects. This study identified the genes for D. citri ecdysone receptor (DcEcR) and ultraspiracle (DcUSP). According to the qPCR data, DcUSP peaked at the 5th-instar nymph stage, while DcEcR peaked at the adult stage. Females expressed DcEcR and DcUSP at much higher levels than males. RNAi was used to examine DcEcR and DcUSP function. The findings demonstrated that inhibition of DcEcR and DcUSP delayed nymph development and decreased survival and eclosion rates. dsEcR caused adults to develop deformed wings, and dsUSP caused nymphs to wither and die. Female adult ovaries developed slowly, and the females laid fewer eggs. Additionally, DcEcR and DcUSP were inhibited, increasing D. citri susceptibility to pesticides. These findings suggest that DcEcR and DcUSP are critical for D. citri development, growth, and reproduction and may serve as potential targets for D. citri management.

Asunto(s)

Citrus , Hemípteros , Insecticidas , Plaguicidas , Receptores de Esteroides , Animales , Femenino , Masculino , Insecticidas/farmacología , Receptores de Esteroides/genética , Hemípteros/fisiología

RESUMEN

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

Asunto(s)

Daphnia , Saccharomyces cerevisiae , Animales , Humanos , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Ligandos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Genes Reporteros , Daphnia/genética , Daphnia/metabolismo , Drosophila melanogaster/genética , Vertebrados/genética , Vertebrados/metabolismo

RESUMEN

Lycosidae females demonstrate meticulous maternal care of offspring by carrying egg sacs and juvenile spiderlings during the reproductive stage. Nuclear receptors (NRs), especially the ecdysone receptor (EcR) and ultraspiracle (USP), have attracted considerable attention in the regulation of arthropod development and reproduction due to their pivotal roles in ecdysteroid signaling cascades. In the present study, 23 NRs, including one EcR and two USPs, were identified in the genome of the predatory wolf spider Pardosa pseudoannulata. RNA interference (RNAi) targeting EcR and USP-1 inhibited spiderling development and resulted in non-viable eggs in the egg sacs. EcR and USP-1 responded to changes in ecdysteroid levels, and interference in ecdysteroid biosynthesis led to similar phenotypes as dsEcR and dsUSP-1 treatments. These findings suggest that EcR/USP-1-mediated ecdysteroid signaling regulates P. pseudoannulata development and reproduction. The P. pseudoannulata females with suppressed ecdysteroid signaling proactively consumed their non-viable egg sacs, resulting in a 7.19 d shorter first reproductive cycle than the controls. Termination of the failed reproductive cycle enabled the spiders to produce a new egg sac more rapidly. This reproductive strategy may partially rescue the reduction in population growth due to non-viable eggs and compensate for the physiological expenditure of wasted maternal care, which would be beneficial for the conservation of P. pseudoannulata populations and their natural control of insect pests.

Asunto(s)

Ecdisteroides , Arañas , Femenino , Animales , Arañas/genética , Reproducción , Transducción de Señal

RESUMEN

Long-term pesticide-driven selection pressure is one of the main causes of insect outbreaks. In this study, we found that low doses of triflumezopyrim could increase the fecundity of white-backed planthoppers (Sogatella furcifera). By continuously screening 20 generations with a low dose of triflumezopyrim, a triflumezopyrim-resistant strain (Tri-strain, resistance ratio = 20.9-fold) was obtained. The average oviposition quantity and longevity of the Tri-strain (208.77 eggs and 21.31 days, respectively) were significantly higher than those of the susceptible strain (Sus-strain) (164.62 eggs and 17.85 days, respectively). To better understand the mechanism underlying the effects on reproduction, we detected the expression levels of several reproduction-related transcription factors in both the Tri- and Sus-strains. Ultraspiracle (USP) was significantly overexpressed in the Tri-strain. Knockdown of USP by RNAi severely inhibited the moulting process of S. furcifera and disrupted the development of female adult ovaries. Among the potential downstream target genes of USP, Kr-h1 (0.19-fold), Cht8 (0.56-fold) and GPCR A22 (0.31-fold) showed downregulated expression after USP-RNAi. In contrast, the expression of EcR (2.55-fold), which forms heterodimers with USP, was significantly upregulated. Furthermore, RNAi was performed on Kr-h1 in the Tri-strain, and the results show that larval moulting and the development of female adult ovaries were inhibited, consistent with the USP-RNAi results in S. furcifera. These results suggest that the transcription factors USP and Kr-h1 play important roles in the reproductive development of S. furcifera, and overexpression of USP and Kr-h1 in the Tri-resistant strain may result in reproductive outbreaks of pests.

Asunto(s)

Hemípteros , Reproducción , Femenino , Animales , Receptores Citoplasmáticos y Nucleares , Brotes de Enfermedades , Factores de Transcripción/genética

RESUMEN

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators.

Asunto(s)

Proteínas de Drosophila , Insecticidas , Receptores de Esteroides , Animales , Humanos , Ecdisona/farmacología , Ecdisona/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Luciferasas/genética , Hormonas Juveniles , Mamíferos/metabolismo

RESUMEN

BACKGROUND: It is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration. METHODS: The full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells. RESULTS: We identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis. CONCLUSIONS: These data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases.

Asunto(s)

Apoptosis/fisiología , Regulación de la Expresión Génica/fisiología , Receptores de Esteroides/metabolismo , Rhipicephalus/metabolismo , Glándulas Salivales/fisiología , Animales , Clonación Molecular , Conducta Alimentaria , Femenino , Células HEK293 , Humanos , Sistemas de Lectura Abierta , Interferencia de ARN , ARN Bicatenario , Receptores de Esteroides/genética

RESUMEN

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20-hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non-drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third-fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.

Asunto(s)

Escarabajos/crecimiento & desarrollo , Escarabajos/genética , Ecdisterona/metabolismo , Receptores de Esteroides/genética , Animales , Escarabajos/metabolismo , Ecdisterona/farmacología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica/genética , Muda/genética , Interferencia de ARN , Receptores de Esteroides/metabolismo , Transducción de Señal

RESUMEN

In Drosophila melanogaster a significant number of heterogenous larval neurons in the central nervous system undergo metamorphosis-associated programmed cell death, termed metamorphoptosis. Interestingly distinct groups of doomed larval neurons are eliminated at different metamorphic phases. Although ecdysone hormonal signaling via nuclear ecdysone receptors (EcRs) is known to orchestrate the neuronal metamorphoptosis, little is known about how this signaling controls such diverse neuronal responses. Crustacean cardioactive peptide (CCAP)-producing neurons in the ventral nerve cord are developmentally programmed to die shortly after adult emergence. In this study, we show that disruption of endogenous EcR function by ectopic expression of dominant negative forms of EcRs (EcRDN) causes premature death of larval CCAP neurons in a caspase-dependent manner. This event is rescued by co-expression of individual EcR isoforms. Furthermore, larval CCAP neurons are largely normal in ecr mutants lacking either EcR-A or EcR-B isoforms, suggesting that EcR isoforms redundantly function to protect larval CCAP neurons. Of surprise, a role of Ultraspiracle (Usp), a canonical partner of EcR, is dispensable in the protection of CCAP neurons, whereas both EcR and Usp are required for inducing metamorphoptosis of vCrz neurons shortly after prepupal formation. As a downstream, grim is an essential cell death gene for the EcRDN-mediated CCAP neuronal death, while either hid or rpr function is dispensable. Together, our results suggest that Usp-independent EcR actions protect CCAP neurons from their premature death by repressing grim expression until their normally scheduled apoptosis at post-emergence. Our studies highlight two opposite roles played by EcR function for metamorphoptosis of two different peptidergic neuronal groups, proapoptotic (vCrz) versus antiapoptotic (CCAP), and propose that distinct death timings of doomed larval neurons are determined by differential signaling mechanisms involving EcR.

Asunto(s)

Apoptosis/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Larva/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Receptores de Esteroides/metabolismo , Animales , Caspasas/metabolismo , Muerte Celular/fisiología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiología

RESUMEN

Nuclear receptors (NRs) are a family of ligand-dependent transcription factors activated by lipophilic compounds. NRs share a common structure comprising three domains: a variable N-terminal domain (NTD), a highly conserved globular DNA-binding domain and a ligand-binding domain. There are numerous papers describing the molecular details of the latter two globular domains. However, very little is known about the structure-function relationship of the NTD, especially as an intrinsically disordered fragment of NRs that may influence the molecular properties and, in turn, the function of globular domains. Here, we investigated whether and how an intrinsically disordered NTD consisting of 58 amino acid residues affects the functions of the globular domains of the Ultraspiracle protein from Helicoverpa armigera (HaUsp). The role of the NTD was examined for two well-known and easily testable NR functions, i.e., interactions with specific DNA sequences and dimerization. Electrophoretic mobility shift assays showed that the intrinsically disordered NTD influences the interaction of HaUsp with specific DNA sequences, apparently by destabilization of HaUsp-DNA complexes. On the other hand, multi-angle light scattering and sedimentation velocity analytical ultracentrifugation revealed that the NTD acts as a structural element that stabilizes HaUsp homodimers. Molecular models based on small-angle X-ray scattering indicate that the intrinsically disordered NTD may exert its effects on the tested HaUsp functions by forming an unexpected scorpion-like structure, in which the NTD bends towards the ligand-binding domain in each subunit of the HaUsp homodimer. This structure may be crucial for specific NTD-dependent regulation of the functions of globular domains in NRs.

Asunto(s)

ADN/química , Proteínas de Insectos/química , Proteínas Intrínsecamente Desordenadas/química , Dominios y Motivos de Interacción de Proteínas , Animales , ADN/metabolismo , Proteínas de Insectos/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Mariposas Nocturnas , Conformación Proteica

RESUMEN

Ecdysteroid hormones are pivotal in the development, growth, and molting of arthropods, and the hormone pathway is triggered by binding ecdysteroid to a heterodimer of the two nuclear receptors; ecdysone receptors (EcR) and ultraspiracle (USP). We have characterized EcR and USP genes, and their 5'-untranslated region (5'-UTR) from the copepod Paracyclopina nana, and studied mRNA transcription levels in post-embryonic stages and in response to water accommodated fractions (WAFs) of crude oil. The open reading frames (ORF) of EcR and USP were 1470 and 1287bp that encoded 490 and 429 amino acids with molecular weight of 121.18 and 105.03kDa, respectively. Also, a well conserved DNA-binding domain (DBD) and ligand-binding domain (LBD) were identified which confirmed by phylogenetic analysis. Messenger RNA transcriptional levels of EcR and USP were developmental stage-specific in early post-embryonic stages (N3-4). However, an evoked expression of USP was observed throughout copepodid stage and in adult females. WAFs (40 and 80%) were acted as an ecdysone agonist in P. nana, and elicited the mRNA transcription levels in adults. Developmental stage-specific transcriptional activation of EcR and USP in response to WAFs was observed. USP gene was down-regulated in the nauplius in response to WAF, whereas up-regulation of USP was observed in the adults. This study represents the first data of molecular elucidation of EcR and USP genes and their regulatory elements from P. nana and the developmental stage specific expression in response to WAFs, which can be used as potential biomarkers for environmental stressors with ecotoxicological evaluations in copepods.

Asunto(s)

Proteínas de Artrópodos/metabolismo , Copépodos/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Contaminantes Químicos del Agua/toxicidad , Regiones no Traducidas 5' , Animales , Proteínas de Artrópodos/genética , Copépodos/genética , Copépodos/metabolismo , Proteínas de Unión al ADN/genética , Monitoreo del Ambiente/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Metamorfosis Biológica , Filogenia , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Elementos Reguladores de la Transcripción , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos

RESUMEN

RNA interference (RNAi) has been widely used in functional genomics of insects and received intensive attention in the development of RNAi-based plants for insect control. Ecdysone receptor (EcR) and ultraspiracle protein (USP) play important roles in molting, metamorphosis, and reproduction of insects. EcR and USP orthologs and their function in grain aphid (Sitobion avenae F.) have not been documented yet. Here, RT-PCR, qRT-PCR, dsRNA feeding assay and aphid bioassay were employed to isolate EcR and USP orthologs in grain aphid, investigate their expression patterns, and evaluate the effect of RNAi on aphid survival and fecundity, and its persistence. The results indicated that SaEcR and SaUSP exhibited similar expression profiles at different developmental stages. Oral administration of dsRNAs of SaEcR and dsSaUSP significantly decreased the survival of aphids due to the down-regulation of these two genes, respectively. The silencing effect was persistent and transgenerational, as demonstrated by the reduced survival and fecundity due to knock-down of SaEcR and SaUSP in both the surviving aphids and their offspring, even after switching to aphid-susceptible wheat plants. Taken together, our results demonstrate that SaEcR and SaUSP are essential genes in aphid growth and development, and could be used as RNAi targets for wheat aphid control.

Asunto(s)

Áfidos/genética , Fertilidad/genética , Genes de Insecto , Herbivoria/genética , Proteínas de Insectos/genética , Interferencia de ARN , Receptores de Esteroides/genética , Triticum/parasitología , Animales , Áfidos/crecimiento & desarrollo , Perfilación de la Expresión Génica , Proteínas de Insectos/metabolismo , Filogenia , ARN Bicatenario/metabolismo , Receptores de Esteroides/metabolismo

RESUMEN

Ecdysteroids play an important role in the growth, development, and reproduction of insects. The ecdysteroid signal is transduced through a heterodimeric complex of two nuclear receptors: the ecdysone receptor (EcR) and the ultraspiracle (USP). To investigate the role of USP during the development of Grapholita molesta, ultraspiracle (USP) cDNA was obtained from the transcriptome of G. molesta and verified by PCR. The deduced amino acid sequence of G. molesta USP (GmUSP) was highly homologous to those of other lepidopterans, especially the Manduca sexta USP-1 isoform. Relatively high expression levels of GmUSP were observed immediately prior to the last larval (L10) molt and during the early pupal stage (P1). Furthermore, high levels of GmUSP mRNA were found in the midgut during the last larval stage. Silencing of GmUSP could significantly down-regulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and a prolonged prepupal duration. In RNAi experiments, ecdysone-inducible genes were significantly down-regulated following GmUSP silencing, suggesting that they were under the control of GmUSP. In addition, the GmUSP knockdown resulted in increased mortality during metamorphosis and prolonged the prepupal period. Taken together, the present results indicate that GmUSP may directly or indirectly affect the development of G. molesta.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/metabolismo , Modelos Moleculares , Mariposas Nocturnas/fisiología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , China , Biología Computacional , Secuencia Conservada , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/química , Proteínas de Insectos/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Datos de Secuencia Molecular , Mariposas Nocturnas/crecimiento & desarrollo , Filogenia , Pupa/crecimiento & desarrollo , Pupa/fisiología , Interferencia de ARN , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/genética

RESUMEN

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval-pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real-time quantitative polymerase chain reaction in different tissues during the larval-pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein-protein interaction were explored by transient expression and far-western blotting, respectively. All the four genes were significantly up-regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20-hydroxyecdysone (20E) induction except for USP2, and USP1 could be up-regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein-protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval-pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.

Asunto(s)

Mariposas Nocturnas/fisiología , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Ecdisterona/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos , Hormonas Juveniles/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica/fisiología , Datos de Secuencia Molecular , Muda/fisiología , Mariposas Nocturnas/genética , Isoformas de Proteínas/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Esteroides/genética , Factores de Transcripción/genética

RESUMEN

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Šresolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.

Asunto(s)

Ischnocera/química , Receptores de Esteroides/metabolismo , Animales , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores de Esteroides/química

RESUMEN

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD-NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.

Asunto(s)

Aedes/química , Bombyx/química , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Drosophila melanogaster/química , Proteínas Intrínsecamente Desordenadas/química , Receptores de Esteroides/química , Factores de Transcripción/química , Aedes/genética , Animales , Bombyx/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Escherichia coli , Hidrodinámica , Proteínas Intrínsecamente Desordenadas/genética , Cinética , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Esteroides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Activación Transcripcional

RESUMEN

Morphogenetic signaling by small terpenoid hormones is a common feature of both vertebrate and invertebrate development. Most attention on insect developmental signaling by small terpenoids has focused on signaling by juvenile hormone through bHLH-PAS proteins (e.g., the MET protein), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a series of endocrine and pharmacological studies on pupariation in cyclorrhaphous Diptera have remained persistently refractory to explanation with the above two-axis model. Recently, the terpenoid compound methyl farnesoate has been physicochemically demonstrated to exist in circulation at physiological concentrations, in several mecopterid orders, including Diptera. In addition, it has also been recently demonstrated that the receptor to which methyl farnesoate binds with nanomolar affinity (ultraspiracle, an ortholog of retinoid X receptor) requires a functioning ligand binding pocket to sustain the morphogenetic transition to puparium formation. This review evaluates endocrine and pharmacological evidence for developmental pathways reached by methyl farnesoate action, and assesses the participation of the retinoid X receptor ligand pocket in signal transduction to those developmental endpoints.

Asunto(s)

Dípteros/metabolismo , Receptores X Retinoide/metabolismo , Sesquiterpenos/metabolismo , Animales , Metamorfosis Biológica/genética , Metamorfosis Biológica/fisiología , Transducción de Señal/fisiología

RESUMEN

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.

Asunto(s)

Bombyx/crecimiento & desarrollo , Hidrazinas/administración & dosificación , Ovario/efectos de los fármacos , Animales , Bombyx/efectos de los fármacos , Ecdisterona/genética , Ecdisterona/metabolismo , Femenino , Hemolinfa/metabolismo , Oogénesis/efectos de los fármacos , Ovario/crecimiento & desarrollo , Vitelogénesis/efectos de los fármacos , Vitelogénesis/genética , Vitelogeninas/genética , Vitelogeninas/metabolismo

RESUMEN

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3-5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5-4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5' UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.

Asunto(s)

Proteínas de Artrópodos/genética , Braquiuros/genética , Muda , Receptores de Esteroides/genética , Receptores X Retinoide/genética , Transcriptoma , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Braquiuros/crecimiento & desarrollo , Braquiuros/metabolismo , Clonación Molecular , Secuencia Conservada , Ecdisteroides/sangre , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismo , Receptores X Retinoide/metabolismo , Análisis de Secuencia de ADN

RESUMEN

The 20-hydroxyecdysone receptor (EcR) is a transcription factor belonging to the nuclear receptor superfamily. Together with the ultraspiracle nuclear receptor (Usp) it coordinates critical biological processes in insects such as development and reproduction. EcR and its ligands are used in commercially available ecdysone-inducible expression systems and are considered to be artificial gene switches with potential therapeutic applications. However, the regulation of EcR action is still unclear, especially in mammals and as far as posttranslational modifications are concerned. Up until now, there has been no study on EcR sumoylation. Using bioinformatic predictors, a Ubc9 fusion-directed sumoylation system and mutagenesis experiments, we present EcR as a new target of SUMO1 and SUMO3 modification. Our research revealed that EcR undergoes isoform-specific multisumoylation. The pattern of modification remains unchanged in the presence of the ligand and the dimerization partner. The SUMO acceptor sites are located in the DNA-binding domain and the ligand-binding domain that both exhibit structural plasticity. We also demonstrated the existence of a sumoylation site in the F region and EcRA-A/B region, both revealing characteristics of intrinsically disordered regions. The consequences of modification and the resulting impact on conformation and function may be especially crucial for the disordered sequences in these two areas. The isoform-specificity of sumoylation may explain the differences in the transcriptional activity of EcR isoforms.

Asunto(s)

Proteínas de Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animales , Western Blotting , Línea Celular , Biología Computacional , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Modelos Biológicos , Receptores de Esteroides/química , Receptores de Esteroides/genética , Sumoilación