Mietek Wozniak

Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle

The identification of potential endogenous or synthetic ligands for orphan receptors in the steroid receptor superfamily is important both for discerning endogenous regulatory pathways and for designing receptor inhibitors. The insect nuclear receptor Ultraspiracle (USP), an ortholog of vertebrate RXR, has long been treated as an orphan receptor. We have tested here the fit of terpenoid ligands to the JH III-binding site of monomeric and homo-oligomeric USP from Drosophila melanogaster (dUSP). dUSP specifically bound juvenile hormone III (JH III), but not control farnesol or JH III acid, and also specifically changed in conformation upon binding of JH III in a fluorescence binding assay. Juvenile hormone III binding caused intramolecular changes in receptor conformation, and stabilized the receptors dimeric/oligomeric quaternary structure. In both a radiometric competition assay and the fluorescence binding assay the synthetic JH III agonist methoprene specifically competed with JH III for binding to dUSP, the first demonstration of specific binding of a biologically active JH III analog to an insect nuclear receptor. The recombinant dUSP bound with specificity to a DR12 hormone response element in a gel shift assay. The same DR12 element conferred enhanced transcriptional responsiveness of a transfected juvenile hormone esterase core promoter to treatment of transfected cells with JH III, but not to treatment with retinoic acid or T3. The activity of JH III or JH III-like structures, but not structures without JH III biological activity, to bind specifically to dUSP and activate its conformational change, provide evidence of a terpenoid endogenous ligand for Ultraspiracle, and offer the prospect that synthetic, terpenoid structures may be discovered that can agonize or antagonize USP function in vivo.

The retinoid-X receptor ortholog, ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand

The in vivo ligand‐binding function and ligand‐binding activity of the Drosophila melanogaster retinoid‐X receptor (RXR) ortholog, ultraspiracle, toward natural farnesoid products of the ring gland were assessed. Using an equilibrium fluorescence‐binding assay, farnesoid products in the juvenile hormone (JH) biosynthesis pathway, and their epoxy derivatives, were measured for their affinity constant for ultraspiracle (USP). Farnesol, farnesal, farnesoic acid and juvenile hormone III exhibited high nanomolar to low micromolar affinity, which in each case decreased upon addition of an epoxide across a double bond of the basic farnesyl structure. Similar analysis of the substitution on C1 of methyl ether, alcohol, aldehyde, and carboxylic acid showed that each conferred weaker affinity than that provided by the methyl ester. Attention was thus focused for a ring‐gland farnesoid product that possesses the features of methyl ester and lack of an epoxide. A secreted product of the ring gland, methyl farnesoate, was identified possessing these features and exhibited an affinity for ultraspiracle (Kd = 40 nm) of similar strength to that of RXR for 9‐cis retinoic acid. Mutational analysis of amino acid residues with side chains extending into the ligand‐binding pocket cavity (and not interacting with secondary receptor structures or extending to the receptor surface to interact with coactivators, corepressors or receptor dimer partners) showed that the mutation C472A/H475L strongly reduced USP binding to this ring gland product and to JH III, with less effect on other ring‐gland farnesoids and little effect on binding by (the unnatural to Drosophila) JH I. Along with the ecdysone receptor, USP is now the second arthropod nuclear hormone receptor for which a secreted product of an endocrine gland that binds the receptor with nanomolar affinity has been identified.

Biochemical and immunological properties of different electrophoretic forms of juvenile hormone esterase from Trichoplusia ni (Hübner)

The two major electrophoretic forms (pI 5.5, 5.3) of juvenile hormone esterase were independently isolated from hemolymph of larval Trichoplusia ni. A simple and rapid preparation procedure of poly(ethylene glycol) precipitation, Sephadex gel filtration and chromatofocusing is described. Analytical isoelectric focusing showed only one peak of juvenile hormone esterase activity in the respective purified samples, whereas there were four (two major) such peaks in the hemolymph. The amino acid composition of the two forms was similar. The comparison of peptides obtained after protein fragmentation by cyanogen bromide showed that juvenile hormone esterases A and B were very similar, although definitely not identical, in amino acid sequence. The immunological comparisons of juvenile hormone esterases suggested that the number of polyclonal antibody binding sites on both forms was the same. There were no detected differences between immunoreactive properties of juvenile hormone esterase from the hemolymph of different stages of larval maturation. The influence of the active site of the enzyme on its antigenic properties was studied by immunocompetition. The inactive, heat-denatured juvenile hormone esterase can only partially protect against inhibition of its activity by the antibodies, whereas an organophosphate inhibitor which covalently binds to the catalytic center of the enzyme did not change the immunoreactive properties in comparison to active juvenile hormone esterase from hemolymph. These data show that heat-denatured juvenile hormone esterase has lost at least one or more epitopes, but the catalytic site of the enzyme is distinct from the epitopes.

Immunochemical characterization of juvenile hormone esterase from different species of lepidoptera

Cross-immunoreactivity of juvenile hormone esterase (JHE) from different species was tested using anti-JHE (Trichoplusia ni) (Noctuidae) polyclonal antibody. Partial cross-reactivity was observed between JHE from Hyphantria cunea, Isia isabella (Arctuidae) and Spodoptera exigua (Noctuidae) in immunoblot analysis. Soluble antigen-antibody complex formation was observed between anti-JHE (T. ni) and antigen(s) from Heliothis virescens (Noctuidae) during immunotitration of antigen(s). Using an ELISA method the highest cross-reactivity was observed for both species from Arctuidae and lower cross-reactivity for antigen(s) from H. virescens.

Regulatory mediators in the venom of Chelonus sp.: Their biosynthesis and subsequent processing in homologous and heterologous systems

Following titration of the contents of the venom gland reservoir, the rate of biosynthesis of venom proteins was sufficiently rapid over the next 6-24 hrs to restore their titer to the level initially synthesized during early adulthood. There was no evidence of processing of smaller molecular weight components from much larger forms. Although most proteins were stable in young host embryos, two specific processing products of a 32.5 kDa venom protein were found in such hosts. The natural injection of venom proteins into either very old embryos or young embryos subsequently held at 4 degrees C for six days resulted in rapid degradation to biologically inactive forms. These data are the first report of direct examination of the biosynthesis of wasp venom proteins and the first analysis of the processing of specific hymenopteran venom proteins in target tissues.

Newly identified, basic hemolymph proteins from noctuid species

A number of basic metamorphosis-associated proteins were identified from several noctuid species. All of these proteins have molecular weights in the range of 73,000 to 74,000. Two of the proteins in Trichoplusia ni and Heliothis virescens were found to be suppressible by a juvenile hormone analog.

Characterization of two major isoforms of juvenile hormone esterase from Trichoplusia ni (Lepidoptera)

The two major isoforms of juvenile hormone (JH) esterase isolated from Trichoplusia ni were fragmented by cyanogen bromide and trypsin digestion. The resulting CNBr or CNBr/trypsin fragments were characterized and compared biochemically by SDS-PAGE, isoelectric focusing, two-dimensional electrophoresis and HPLC. Similar and unique fragments were examined for sequence, antigenic determinants and carbohydrate moieties. The studies identified small regions of the proteins which possess either potentially different sequences or different post-translational modifications. The location of a glycosylated asparagine residue was determined, as well as a region containing an epitope probably composed of a linear sequence of residues. An N-terminal region was identified that contained charge variation between the two isoforms and the sequence was obtained for the only unique CNBr/trypsin fragment detected from that region. These are the first data on mapping of regions of charge variation, epitope location and glycosylation sites for this enzyme from any insect species.

Glycosylation and isoform variation of juvenile hormone esterase in the fat body and hemolymph during metamorphosis of Trichoplusia ni (Lepidoptera).

Two major isoforms of juvenile hormone esterase (JH esterase) from metamorphosing larvae of Trichoplusia ni were characterized with respect to isoform variation, glycosylation (lectin reactivity) and hormonal induction. Both forms are similarly inducible by juvenile hormone, and both become similarly glycosylated, as measured by concanavalin A binding. In prepupae, synthesis of new JH esterase from a low baseline and glycosylation within fat body are limiting steps in modulation of the level of JH esterase in the hemolymph. In contrast, during the pupal stage regulation of hemolymph JH esterase activity changes to a level other than synthesis of new enzyme or its glycosylation.