Agonist-dependent action of the juvenile hormone receptor.

Juvenile hormone (JH) signaling is effected at the gene regulatory level by receptors of the bHLH-PAS transcription factor family. The sesquiterpenoid hormones and their synthetic mimics are agonist ligands of a unique JH receptor (JHR) protein, methoprene-tolerant (MET). Upon binding an agonist to its PAS-B cavity, MET dissociates from a cytoplasmic chaperone complex including HSP83 and concomitantly switches to a bHLH-PAS partner taiman, forming a nuclear, transcriptionally active JHR heterodimer. This course of events resembles the vertebrate aryl hydrocarbon receptor (AHR), activated by a plethora of endogenous and synthetic compounds. Like in AHR, the pliable PAS-B cavity of MET adjusts to diverse ligands and binds them through similar mechanisms. Despite recent progress, we only begin to discern agonist-induced conformational shifts within the PAS-B domain, with the ultimate goal to understand how these localized changes stimulate assembly of the active JHR complex, and thus fully grasp the mechanism of JHR signaling.

Frantisek Sehnal: A project that worked out.

Frantisek Sehnal was a prominent and inspiring figure in many areas of insect science, most notably endocrinology, developmental biology, silk research, and recently insect interactions with genetically modified crops. In this article, I will briefly overview Sehnal's research and other academic and educational activities. I would also like to share my personal experience with Frantisek Sehnal as a mentor who drafted, in 1990, a plan for my doctoral thesis: to identify a receptor for juvenile hormone. The project ended up taking more than two decades to complete. While Frantisek has passed away, his legacy stays.

Ligand-dependent protein interactions of the juvenile hormone receptor captured in real time.

Juvenile hormone (JH) signalling provides vital regulatory functions during insect development via transcriptional regulation of genes critical for the progression of metamorphosis and oogenesis. Despite the importance of JH signalling, the underlying molecular mechanisms remain largely unknown. Our current understanding of the pathway depends on static end-point information and suffers from the lack of time-resolved data. Here, we have addressed the dynamic aspect of JH signalling by monitoring in real time the interactions of insect JH receptor proteins. Use of two tags that reconstitute a functional luciferase when in proximity enabled us to follow the rapid assembly of a JH receptor heterodimer from basic helix-loop-helix/Per-Arnt-SIM (bHLH-PAS) proteins, methoprene-tolerant (Met) and taiman (Tai), upon specific JH binding to Met. On a similar timescale (minutes), the dissociation of Met-Met complexes occurred, again strictly dependent on Met interaction with specific agonist ligands. To resolve questions regarding the regulatory role of the chaperone Hsp90/83 in the JHR complex formation, we used the same technique to demonstrate that the Met-Hsp83 complex persisted in the agonist absence but readily dissociated upon specific binding of JH to Met. Preincubation with the Hsp90 inhibitor geldanamycin showed that the chaperone interaction protected Met from degradation and was critical for Met to produce the active signalling dimer with Tai. Thus, the JH receptor functions appear to be governed by principles similar to those regulating the aryl hydrocarbon receptor, the closest vertebrate homologue of the arthropod JH receptor.

Unique peptidic agonists of a juvenile hormone receptor with species-specific effects on insect development and reproduction.

Juvenile hormones (JHs) control insect metamorphosis and reproduction. JHs act through a receptor complex consisting of methoprene-tolerant (Met) and taiman (Tai) proteins to induce transcription of specific genes. Among chemically diverse synthetic JH mimics (juvenoids), some of which serve as insecticides, unique peptidic juvenoids stand out as being highly potent yet exquisitely selective to a specific family of true bugs. Their mode of action is unknown. Here we demonstrate that, like established JH receptor agonists, peptidic juvenoids act upon the JHR Met to halt metamorphosis in larvae of the linden bug, Pyrrhocoris apterus. Peptidic juvenoids induced ligand-dependent dimerization between Met and Tai proteins from P. apterus but, consistent with their selectivity, not from other insects. A cell-based split-luciferase system revealed that the Met-Tai complex assembled within minutes of agonist presence. To explore the potential of juvenoid peptides, we synthesized 120 new derivatives and tested them in Met-Tai interaction assays. While many substituents led to loss of activity, improved derivatives active at sub-nanomolar range outperformed hitherto existing peptidic and classical juvenoids including fenoxycarb. Their potency in inducing Met-Tai interaction corresponded with the capacity to block metamorphosis in P. apterus larvae and to stimulate oogenesis in reproductively arrested adult females. Molecular modeling demonstrated that the high potency correlates with high affinity. This is a result of malleability of the ligand-binding pocket of P. apterus Met that allows larger peptidic ligands to maximize their contact surface. Our data establish peptidic juvenoids as highly potent and species-selective novel JHR agonists.

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation.

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Binding of de novo synthesized radiolabeled juvenile hormone (JH III) by JH receptors from the Cuban subterranean termite Prorhinotermes simplex and the German cockroach Blattella germanica.

Juvenile hormone (JH) controls insect reproduction and development through an intracellular receptor complex comprising two bHLH-PAS proteins, the JH-binding Methoprene-tolerant (Met) and its partner Taiman (Tai). Many hemimetabolous insects including cockroaches strictly depend on JH for stimulation of vitellogenesis. In termites, the eusocial hemimetabolans, JH also regulates the development of caste polyphenism. Studies addressing the agonist ligand binding to recombinant JH receptors currently include three species belonging to two holometabolous insect orders, but none that would represent any of the hemimetabolous orders. Here, we examined JH receptors in two representatives of Blattodea, the cockroach Blattella germanica and the termite Prorhinotermes simplex. To test the JH-binding capacity of Met proteins from these species, we performed chemical synthesis and tritium labeling of the natural blattodean JH homolog, JH III. Our improved protocol increased the yield and specific activity of [10-3H]JH III relative to formerly available preparations. Met proteins from both species specifically bound [3H]JH III with high affinity, whereas Met variants mutated at a critical position within the ligand-binding domain were incapable of such binding. Furthermore, JH III and the synthetic JH mimic fenoxycarb stimulated dimerization between Met and Tai components of the respective JH receptors of both species. These data present primary evidence for agonist binding by JH receptors in any hemimetabolous species and provide a molecular basis for JH action in cockroaches and termites.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness.

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

New ways and new hopes for IGR development.

Insect Growth Regulators (IGRs) represent advanced, bio-rational insecticides. This Special Issue reflects progress in IGR development that has been enabled by insight into the molecular principles of biosynthetic or hormone signaling pathways. The unifying principle is aiming at processes and molecular targets that are unique to arthropods and ideally to narrower insect taxa representing pests or disease vectors. While some strategies of obtaining the desired compounds for chemical intervention rely on rational, structure-based design or computational power, others exploit technologies allowing automated, high-throughput screening of large chemical libraries. All avenues leading to selective and environmentally safe pest control are valid as we face the imminent threat of the declining world insect population.

The juvenile hormone receptor as a target of juvenoid "insect growth regulators".

Synthetic compounds that mimic the action of juvenile hormones (JHs) are founding members of a class of insecticides called insect growth regulators (IGRs). Like JHs, these juvenoids block metamorphosis of insect larvae to reproductive adults. Many biologically active juvenoids deviate in their chemical structure considerably from the sesquiterpenoid JHs, raising questions about the mode of action of such JH mimics. Despite the early deployment of juvenoid IGRs in the mid-1970s, their molecular effect could not be understood until recent discoveries of JH signaling through an intracellular JH receptor, namely the ligand-binding transcription factor Methoprene-tolerant (Met). Here, we briefly overview evidence defining three widely employed and chemically distinct juvenoid IGRs (methoprene, pyriproxyfen, and fenoxycarb), as agonist ligands of the JH receptor. We stress that knowledge of the target molecule is critical for using these compounds both as insecticides and as research tools.

Drosophila macrophages switch to aerobic glycolysis to mount effective antibacterial defense.

Macrophage-mediated phagocytosis and cytokine production represent the front lines of resistance to bacterial invaders. A key feature of this pro-inflammatory response in mammals is the complex remodeling of cellular metabolism towards aerobic glycolysis. Although the function of bactericidal macrophages is highly conserved, the metabolic remodeling of insect macrophages remains poorly understood. Here, we used adults of the fruit fly Drosophila melanogaster to investigate the metabolic changes that occur in macrophages during the acute and resolution phases of Streptococcus-induced sepsis. Our studies revealed that orthologs of Hypoxia inducible factor 1α (HIF1α) and Lactate dehydrogenase (LDH) are required for macrophage activation, their bactericidal function, and resistance to infection, thus documenting the conservation of this cellular response between insects and mammals. Further, we show that macrophages employing aerobic glycolysis induce changes in systemic metabolism that are necessary to meet the biosynthetic and energetic demands of their function and resistance to bacterial infection.

Yeast glucan particles enable intracellular protein delivery in Drosophila without compromising the immune system.

Glucan particles derived from yeast have been recently proposed as potential drug delivery carriers. Here, we demonstrate the potential of glucan particles for protein delivery in vivo, using the insect Drosophila melanogaster as a model organism. By employing genetic tools, we demonstrate the capacity of yeast glucan particles to spread efficiently through the Drosophila body, to enter macrophages and to deliver an active transcription factor protein successfully. Moreover, the glucan particles were nontoxic and induced only minimal immune response. The injection of glucan particles did not impair the ability of Drosophila to fight and survive infection by pathogenic bacteria. From this study, Drosophila emerges as an excellent model to test and develop drug delivery systems based on glucan particles, specifically aimed to regulate macrophages.

Where did the pupa come from? The timing of juvenile hormone signalling supports homology between stages of hemimetabolous and holometabolous insects.

Insect metamorphosis boasts spectacular cases of postembryonic development when juveniles undergo massive morphogenesis before attaining the adult form and function; in moths or flies the larvae do not even remotely resemble their adult parents. A selective advantage of complete metamorphosis (holometaboly) is that within one species the two forms with different lifestyles can exploit diverse habitats. It was the environmental adaptation and specialization of larvae, primarily the delay and internalization of wing development, that eventually required an intermediate stage that we call a pupa. It is a long-held and parsimonious hypothesis that the holometabolous pupa evolved through modification of a final juvenile stage of an ancestor developing through incomplete metamorphosis (hemimetaboly). Alternative hypotheses see the pupa as an equivalent of all hemimetabolous moulting cycles (instars) collapsed into one, and consider any preceding holometabolous larval instars free-living embryos stalled in development. Discoveries on juvenile hormone signalling that controls metamorphosis grant new support to the former hypothesis deriving the pupa from a final pre-adult stage. The timing of expression of genes that repress and promote adult development downstream of hormonal signals supports homology between postembryonic stages of hemimetabolous and holometabolous insects. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Exquisite ligand stereoselectivity of a Drosophila juvenile hormone receptor contrasts with its broad agonist repertoire.

The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)/PAS proteins in Drosophila melanogaster known as germ cell-expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine which structural features define an active JH receptor agonist, we tested several native JHs and their nonnative geometric and optical isomers for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. Our results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand-binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce toward the natural hormone contrasts with the high potency of synthetic Gce agonists of disparate chemistries.

Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture.

Interplay between apicobasal cell polarity modules and the cytoskeleton is critical for differentiation and integrity of epithelia. However, this coordination is poorly understood at the level of gene regulation by transcription factors. Here, we establish the Drosophila activating transcription factor 3 (atf3) as a cell polarity response gene acting downstream of the membrane-associated Scribble polarity complex. Loss of the tumor suppressors Scribble or Dlg1 induces atf3 expression via aPKC but independent of Jun-N-terminal kinase (JNK) signaling. Strikingly, removal of Atf3 from Dlg1 deficient cells restores polarized cytoarchitecture, levels and distribution of endosomal trafficking machinery, and differentiation. Conversely, excess Atf3 alters microtubule network, vesicular trafficking and the partition of polarity proteins along the apicobasal axis. Genomic and genetic approaches implicate Atf3 as a regulator of cytoskeleton organization and function, and identify Lamin C as one of its bona fide target genes. By affecting structural features and cell morphology, Atf3 functions in a manner distinct from other transcription factors operating downstream of disrupted cell polarity.

Genetic Evidence for Function of the bHLH-PAS Protein Gce/Met As a Juvenile Hormone Receptor.

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. Here, we show that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone's vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology.

Molecular basis of juvenile hormone signaling.

Despite important roles played by juvenile hormone (JH) in insects, the mechanisms underlying its action were until recently unknown. A breakthrough has been the demonstration that the bHLH-PAS protein Met is an intracellular receptor for JH. Binding of JH to Met triggers dimerization of Met with its partner protein Tai, and the resulting complex induces transcription of target genes. In addition, JH can potentiate this response by phosphorylating Met and Tai via cell membrane, second-messenger signaling. An important gene induced by the JH-Met-Tai complex is Kr-h1, which inhibits metamorphosis. Kr-h1 represses an 'adult specifier' gene E93. The action of this JH-activated pathway in maintaining the juvenile status is dispensable during early postembryonic development when larvae/nymphs lack competence to metamorphose.

Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing.

The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose.

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.