Analysis of chemosensory genes in Semiothisa cinerearia reveals sex-specific contributions for type-II sex pheromone chemosensation.

Insects employ a sensitive chemosensory system to accurately recognize external odorants, which help them to make a behavioral response quickly. Semiothisa cinerearia has caused serious damages to Sophora japonica L. in recent years, and there is still a lack of effective strategy to control the pest. Although the two type-II sex pheromones of S. cinerearia, 6Z,9Z-cis-3,4-epoxy-17:H and 3Z,6Z,9Z-17:H, have been identified for 30 years, the molecular mechanisms underlying the chemosensation of the two sex pheromones are still unknown. Here, we found that there are differences in the types of antennae sensilla between sexes, and revealed 146 putative chemosensory genes in the antennal transcriptome. Among these genes, 11 and 40 of them displayed male-biased and female-biased expression, respectively. Our findings greatly improve the chemosensory gene resources for S. cinerearia and provide a foundation for functional studies of these sex-biased genes on the chemosensation of sex pheromones and on other sex-related behaviors.

Isolation and characterization of Xenopus soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

High-resolution structure and biochemical properties of the LH1-RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum.

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and ß-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/ß1- or α1/ß3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1-RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.

Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion.

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.

Functional differentiation of two general-odorant binding proteins in Hyphantria cunea (Drury) (Lepidoptera: Erebidae).

BACKGROUND: General odor-binding proteins (GOBPs) play critical roles in insect olfactory recognition of sex pheromones and plant volatiles. Therefore, the identification of GOBPs in Hyphantria cunea (Drury) based on their characterization to pheromone components and plant volatiles is remain unknown. RESULTS: In this study, two H. cunea (HcunGOBPs) genes were cloned, and their expression profiles and odorant binding characteristics were systematically analyzed. Firstly, the tissue expression study showed that both HcunGOBP1 and HcunGOBP2 were highly expressed in the antennae of both sexes, indicating their potential involvement in the perception of sex pheromones. Secondly, these two HcunGOBPs genes were expressed in Escherichia coli and ligand binding assays were used to assess the binding affinities to its sex pheromone components including two aldehydes and two epoxides, and some plant volatiles. HcunGOBP2 showed high binding affinities to two aldehyde components (Z9, Z12, Z15-18Ald and Z9, Z12-18Ald), and showed low binding affinities to two epoxide components (1, Z3, Z6-9S, 10R-epoxy-21Hy and Z3, Z6-9S, 10R-epoxy-21Hy), whereas HcunGOBP1 showed weak but significant binding to all four sex pheromone components. Furthermore, both HcunGOBPs demonstrated variable binding affinities to the plant volatiles tested. Thirdly, in silico studies of HcunGOBPs utilized homology, structure modeling, and molecular docking revealed critical hydrophobic residues might be involved in the binding of HcunGOBPs to their sex pheromone components and plant volatiles. CONCLUSION: Our study suggests that these two HcunGOBPs may serve as potential targets for future studies of HcunGOBPs ligand binding, providing insight in the mechanism of olfaction in H. cunea. © 2023 Society of Chemical Industry.

Allosteric activation of preformed EGF receptor dimers by a single ligand binding event.

Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor in vivo. Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Functional Disparity of Three Pheromone-Binding Proteins to Different Sex Pheromone Components in Hyphantria cunea (Drury).

Hyphantria cunea (Drury) is a destructive invasive pest species in China that uses type II sex pheromone components. To date, however, the binding mechanisms of its sex pheromone components to their respective pheromone-binding proteins (HcunPBPs 1/2/3) have not been explored. In the current study, all three HcunPBPs were expressed in the antennae of both sexes. The prokaryotic expression and ligand binding assays were employed to study the binding of the moth's four sex pheromone components, including two aldehydes and two epoxides, and 24 plant volatiles to the HcunPBPs. Our results showed that the abilities of these HcunPBPs to bind to the aldehydes were significantly different from binding to the epoxides. These three HcunPBPs also selectively bind to some of the plant volatiles tested. Our molecular docking results indicated that some crucial hydrophobic residues might play a role in the binding of HcunPBPs to their sex pheromone components. Three HcunPBPs have different selectivities for pheromone components with both major and minor structural differences. Our study provides a fundamental insight into the olfactory mechanism of moths at the molecular level, especially for moth species that use various type II pheromone components.

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The "Rotation Model".

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in the reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the "rotation model"). This model is also able to explain how oncogenic mutations activate the receptor in the absence of the ligand, without assuming that the mutations induce receptor dimerization. In this review, we discuss the mechanisms underlying the ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model.

The metabolism of lysophosphatidic acids by allelic variants of human soluble epoxide hydrolase.

Lysophosphatidic acids (LPAs) are phospholipids which have many physiological and pathophysiological functions. The human soluble epoxide hydrolase (sEH) plays a role in the metabolism of xenobiotics through its metabolism of aromatic hydrocarbon epoxides such as styrene oxide. sEH also has a phosphatase activity, and metabolizes LPAs. In this study, we investigated a purified wild-type (WT) and six allelic variants of sEH to evaluate differences in their activities toward LPAs. We found that the R103C and R287Q showed significantly lower activity than the WT sEH. We also found that the R103C and R287Q had significantly lower activity even when applied to only the N-terminal or C-terminal domain. The kinetic study determined that the R103C and R287Q had a lower Vmax/Km ratio toward stearoyl-LPA than the other variants. In a previous study, we found that WT sEH suppressed vascular endothelial growth factor (VEGF) mRNA in Hep3B cells; in the present experiments, all sEH variants except V442A suppressed VEGF mRNA levels in Hep3B cells. These results suggest that the R103C and R287Q have lower phosphatase activity, but that all the allelic variants have similar effects on VEGF suppression.