Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

Functional characterization of a special dicistronic transcription unit encoding histone methyltransferase su(var)3-9 and translation regulator eIF2γ in Tribolium castaneum

Operons are rare in eukaryotes, where they often allow concerted expression of functionally related genes. While a dicistronic transcription unit encoding two unrelated genes, the suppressor of position-effect variegation su(var)3-9 and the gamma subunit of eukaryotic translation initiation factor 2 (eIF2γ) has been found in insecta, and its significance is not well understood. Here, we analyzed the evolutionary history of this transcription unit in arthropods and its functions by using model Coleoptera insect Tribolium castaneum. In T. castaneum, Tcsu(var)3-9 fused into the 80 N-terminal amino acids of TceIF2γ, the transcription of these two genes are resolved by alternative splicing. Phylogenetic analysis supports the natural gene fusion of su(var)3-9 and eIF2γ occurred in the ancestral line of winged insects and silverfish, but with frequent re-fission during the evolution of insects. Functional analysis by using RNAi for these two genes revealed that gene fusion did not invoke novel functions for the gene products. As a histone methyltransferase, Tcsu(var)3-9 is primarily responsible for H3K9 di-, and tri-methylation and plays important roles in metamorphosis and embryogenesis in T. castaneum. While TceIF2γ plays essential roles in T. castaneum by positively regulating protein translation mediated ecdysteroid biosynthesis. The vulnerability of the gene fusion and totally different role of su(var)3-9 and eIF2γ in T. castaneum confirm this gene fusion is a non-selected, constructive neutral evolution event in insect. Moreover, the positive relationship between protein translation and ecdysteroid biosynthesis gives new insights into correlations between translation regulation and hormonal signaling.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17β-estradiol, a DmNobo inhibitor. 17β-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17β-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17β-estradiol–mediated inhibition of DmNobo enzymatic activity, as 17β-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

An integrated approach unravels a crucial structural property for the function of the insect steroidogenic Halloween protein Noppera-bo

Ecdysteroids are the principal insect steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis, encoded by Halloween genes, have been identified and well characterized, both genetically and biochemically. However, none of these proteins have yet been characterized at the tertiary structure level. Here, we report an integrated in silico, in vitro, and in vivo analyses of the Halloween glutathione S-transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with glutathione and 17β-estradiol, a DmNobo inhibitor. 17β-estradiol almost fully occupied the putative ligand-binding pocket, and a prominent hydrogen bond formed between Asp113 of DmNobo and 17β-estradiol. Asp113 is essential for inhibiting DmNobo enzymatic activity by 17β-estradiol, as 17β-estradiol does not inhibit and physically interacts less with the Asp113Ala DmNobo point mutant. Asp113 is highly conserved among Nobo proteins, but not among other GSTs, implying that Asp113 is important for endogenous Nobo function. Indeed, a homozygous nobo allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue, which seems to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. This is the first study to reveal the structural characteristics of insect steroidogenic Halloween proteins. This study also provides basic insight into applied entomology for developing a new type of insecticides that specifically inhibit ecdysteroid biosynthesis.Significance StatementInsect molting and metamorphosis are drastic and dynamic biological processes and, therefore, have fascinated many scientists. Ecdysteroids represent one class of insect hormones that are indispensable for inducing molting and metamorphosis. It is well known that proteins responsible for catalyzing ecdysteroid biosynthesis reactions are encoded by “Halloween” genes, most of which have names of ghosts and phantoms. However, no studies have focused on the structural properties of these biosynthetic proteins. In this study, we addressed this unsolved issue and successfully unraveled a structural property that is crucial for the function of the fruit fly Halloween protein, Noppera-bo (a Japanese faceless ghost). This is the first study to reveal the structural characteristics of an insect steroidogenic Halloween protein.