Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti

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.     

RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica

Ecdysteroids play a major role during developmental growth in insects. The more active form of these hormones, 20-hydroxyecdysone (20E), acts upon binding to its heterodimeric receptor, formed by the two nuclear receptors, EcR and RXR/USP. Functional characterization of USP has been exclusively conducted on the holometabolous insect Drosophila melanogaster. However, it has been impossible to extend such analysis to primitive-hemimetabolous insects since species of this group are not amenable to genetic analysis. The development of methodologies based on gene silencing using RNA interference (RNAi) after treatment with double-stranded RNA (dsRNA) in vivo has resolved such limitations. In this paper, we show that injection of dsRNA into the haemocoel of nymphs and adults of the cockroach Blattella germanica can be used to silence gene function in vivo. In our initial attempt to test RNAi techniques, we halted the expression of the adult-specific vitellogenin gene. We then used the same technique to silence the expression of the B. germanica RXR/USP (BgRXR) gene in vivo during the last nymphal instar. BgRXR knockdown nymphs progressed through the instar correctly but they arrested development at the end of the stage and were unable to molt into adults. The results described herein suggest that RXR/USP function, in relation to molting, is conserved across the insect Class.     

The structure of the USP/RXR of <Emphasis Type="Italic">Xenos pecki</Emphasis> indicates that Strepsiptera are not closely related to Diptera

The receptor for the insect molting hormone, ecdysone, is a heterodimer consisting of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The ligand binding domain sequences of arthropod USPs divide into two distinct groups. One group consists of sequences from members of the holometabolous Lepidoptera and Diptera, while the other arthropod sequences group with vertebrate retinoid-X-receptors (RXRs). We therefore wondered whether USP/RXR structure could be used to clarify the contentious phylogenetic position of the order Strepsiptera, which has proposed affinities with either Diptera or Coleoptera. We have cloned and sequenced the USP/RXR from the strepsipteran Xenos pecki. Phylogenetic analyses are not consistent with a close affinity between Strepsiptera and Diptera.Electronic Supplementary Material Supplementary material is available for this article at Edited by D. Tautz     

Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR)

The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the complex formation of ligand-binding domains EcR and USP ligand is different.     

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.