Odorant-binding Proteins Research Articles

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae).

Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (RPL32, RPS23, SDHA, UBQ, UCCR, GST, TATA-box, HSP70, GAPDH, RPL7A, SOD, RPS3, Actin, α-tubulin, AK, and EF1) were carefully chosen in R. pedestris, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in R. pedestris is contingent upon the specific experimental conditions. RPL7A and EF1 are optimal reference genes for developmental stages. Furthermore, α-tubulin and EF1 exhibit the most stable expression across various adult tissues. RPL32 and RPL7A exhibit the most stable expression for adult age. For nymph age, RPL32 and SOD display the most stable expression. For temperature conditions, RPS23 and RPL7A were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (RpedOBP37) and cytochrome P450 6a2 (RpedCYP6) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on R. pedestris, serving as a fundamental groundwork for future investigations on stable reference genes in R. pedestris as well as other organisms.

Binding Properties of Odorant-Binding Protein 7 to Host Volatiles in Larvae of Spodoptera frugiperda.

The chemosensory system is crucial during the growth and development of the moths. Spodoptera frugiperda (Lepidoptera: Noctuidae) is one of the most destructive insect pests. However, there is little functional research on odorant-binding proteins (OBPs) in the larval stage of S. frugiperda. Here, we obtained SfruOBP7 from transcriptomics and conducted the sequence analysis. We used quantitative real-time PCR to explore the expression profiles of SfruOBP7. The function identification showed that SfruOBP7 has a binding ability to 18 plant volatiles. Further molecular docking and site-directed mutant assay revealed that Lys45 and Phe110 were the key binding sites for SfruOBP7 interacting with linalool. In the behavior assays, linalool could attract the larvae, and dsOBP7-treated larvae lost their attraction to linalool. Our results help to reveal the essential molecular mechanism of the olfactory perception in the larvae and design an attractant based on the host volatiles.

The antennal transcriptome analysis and characterizations of odorant-binding proteins in Megachile saussurei (Hymenoptera, Megachilidae)

BackgroundOdorant-binding proteins (OBPs) are essential in insect’s daily behaviors mediated by olfactory perception. Megachile saussurei Radoszkowski (Hymenoptera, Megachilidae) is a principal insect pollinating alfalfa (Medicago sativa) in Northwestern China. The olfactory function have been less conducted, which provides a lot of possibilities for our research.ResultsOur results showed that 20 OBPs were identified in total. Multiple sequence alignment analysis indicated MsauOBPs were highly conserved with a 6-cysteine motif pattern and all belonged to the classic subfamily, coding 113-196 amino acids and sharing 41.32%-99.12% amino acid identity with known OBPs of other bees. Phylogenetic analysis indicated there were certain homologies existed among MsauOBPs and most sequences were clustered with that of Osmia cornuta (Hymenoptera, Megachilidae). Expression analysis showed the identified OBPs were mostly enriched in antennae instead of other four body parts, especially the MsauOBP2, MsauOBP3, MsauOBP4, MsauOBP8, MsauOBP11 and MsauOBP17, in which the MsauOBP2, MsauOBP4 and MsauOBP8 presented obvious tissue-biased expression pattern. Molecular docking results indicated MsauOBP4 might be the most significant protein in recognizing alfalfa flower volatile 3-Octanone, while MsauOBP13 might be the most crucial protein identifying (Z)-3-hexenyl acetate. It was also found the lysine was a momentous hydrophilic amino acid in docking simulations.ConclusionIn this study, we identified and analyzed 20 OBPs of M. saussurei. The certain homology existed among these OBPs, while some degree of divergence could also be noticed, indicating the complex functions that different MsauOBPs performed. Besides, the M. saussurei and Osmia cornuta were very likely to share similar physiological functions as most of their OBPs were clustered together. MsauOBP4 might be the key protein in recognizing 3-Octanone, while MsauOBP13 might be the key protein in binding (Z)-3-hexenyl acetate. These two proteins might contribute to the alfalfa-locating during the pollination process. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species.

An Antennae-Enriched Odorant-Binding Protein EonuOBP43 Mediate the Behavioral Response of the Tea Green Leafhopper, Empoasca onukii Matsuda to the Host and Nonhost Volatiles.

Olfaction is crucial for Empoasca onukii Matsuda to recognize odors from the host and nonhost plants, and it has been proposed that odorant binding proteins are directly required for odorant discrimination and represent potential targets of interest for pest control. Here, we cloned EonuOBP43 and expressed the recombinant EonuOBP43 protein. Furthermore, competitive fluorescence binding assays with 19 ligands indicated that terpenoids and alkanes showed a relatively higher than for other classes of chemicals. Additionally, ligand docking and site-directed mutagenesis results revealed that seven hydrophobic residues, including Val-86, Met-89, Phe-90, Ile-104, Ile-105, Leu-130, and Val-134, played a key role in the binding of EonuOBP43 to plant volatiles. In olfactometer tests, E. onukii were significantly attracted to α-farnesene and repelled to β-caryophyllene, and dsOBP43 treated adult lost response to α-farnesene and β-caryophyllene. In summary, our results demonstrated that EonuOBP43 may function as a carrier in the process of sensing plant compounds of E. onukii.

Isolation, characterization and docking analysis of insecticidal compound from Ocimum canum methanolic leaf extracts and its potential against three mosquito vectors

Vector management is an important challenge and burden to developing countries. Frequent and indiscriminate application of chemical insecticides for vector control has resulted in the development of resistance and undesirable effects on beneficial organisms. Hence an alternative approach is needed for the mosquito control programme. The present study investigated the insecticidal activity and spectral analysis of Ocimum canum methanol leaf extract against three mosquito species. One out of four fractions (F1-F4), the 4th fraction, revealed showed good insecticidal activity in larva and adult Cx. quinquefasciatus, Ae. aegypti and An. stephensi (0.114, 0.325 and 0.173 ppm) and (1.798, 1.061 and 0.871 ppm). The mortality was found to be dosage dependant. Spectral analysis of 1H NMR and 13C NMR indicates the presence of 6 protons at δH value of 7.45 (s) and 3 protons at δH value of 7.85 (s) ppm. FT-IR identified the possible band near the capping region at 3417.16 cm-1. LC-MS confirmed the eugenol compound with 99.79% purity at the molecular weight of 164. Further, eugenol docking analysis revealed high homology similarity with insect odorant binding protein 3OGN and AchE protein 2BG9. Overall, the result suggests that isolated eugenol compounds were found to be effective for the eco-friendly management of mosquito vectors. These findings could be useful for designing an efficient mosquitocidal compound in the future.

A Study of Adult Olfactory Proteins of Primitive Ghost Moth, Endoclita signifer (Lepidoptera, Hepialidae).

Endoclita signifer is a prominent wood-boring insect species in eucalyptus plantations in Guangxi, China, causing significant ecological and economic damage. A novel approach to controlling the challenging wood-boring pest involves disrupting the olfactory communication between insects and the volatile compounds emitted by plants. To identify the olfactory proteins contributing to host selection based on 11 GC-EAD-active volatiles from eucalyptus leaves and to discover the highly expressed olfactory proteins, we conducted a study on the antennal transcriptomes of adult E. signifer and screened key olfactory proteins in the antennae. We identified a total of 69 olfactory proteins. When compared to the larval transcriptomes, the antennal transcriptome of adult E. signifer revealed the presence of 17 new odorant-binding proteins (OBPs), including 2 pheromone-binding proteins (PBPs), 7 previously unreported chemosensory proteins (CSPs), 17 new odorant receptors (ORs), 4 new gustatory receptors (GRs), 11 novel ionotropic receptors (IRs), and 2 sensory neuron membrane proteins (SNMPs). Through the phylogenetic tree of OBPs and ORs, we identified EsigPBP2 and EsigPBP3 as two of the three PBPs, designated EsigOR13 as EsigOrco, and recognized EsigOR10 and EsigOR22 as the newly discovered EsigPRs in E. signifer. In the adult antennae, the expression levels of EsigGOBP14, EsigGOBP13, EsigOBP14, EsigOBP17, EsigCSP14, and EsigOR16 were notably high, indicating that these proteins could be pivotal in binding to plant volatiles.

The essential oil of Kochia scoparia (L.) Schrad. as a potential repellent against stored-product insects.

Chemical composition of the essential oil from Kochia scoparia (L.) Schrad. (syn. Bassia scoparia (L.) A. J. Scott) was analyzed in quality and quantity by GC-MS and GC-FID. Repellent activities of the essential oil from K. scoparia (KSEO) were evaluated against two common species of stored-product insects Tribolium castaneum Herbst and Liposcelis bostrychophila Badonnel. Results indicated that KSEO mainly consisted of eugenol, β-caryophyllene, and α-humulene, accounting for 75.6%, 8.2%, and 1.4% of the total oil, respectively. KSEO and the three major components were repellent to T. castaneum and L. bostrychophila adults. Notably, KSEO exerted significant effects, comparable to the positive control DEET at 2 and 4h post-exposure. Eugenol at 63.17-2.53 nL/cm2 exhibited high percentage repellency ranging from 96 to 70% against L. bostrychophila during 4-h exposure. To gain further insights into the repellent activity, molecular docking simulation was performed with eugenol as the ligand and an odorant binding protein TcOBPC12 (gene: TcOBP10B) from the model insect T. castaneum as the receptor. Docking calculation results revealed that TcOBPC12 had binding affinity to eugenol (△G = - 4.52kcal/mol) along with a hydrogen bond of 0.18nm (1.8Å) long forming between them, which could be an important target protein associated with identifying volatile repellent molecules. This work highlights the promising potential of KSEO as a botanical repellent for controlling stored-product insects.

Insights on reproduction-related genes in the striped fruit fly, Zeugodacus scutellata (Hendel) (Diptera: Tephritidae).

The striped fruit fly, Zeugodacus scutellata is a significant pest in East and Southeast Asia by damaging Cucurbitaceae blossoms and fruits. To control this pest, a novel strategy to suppress the gene(s) associated with sexually dimorphic phenotypes has been devised and implemented in a laboratory scale. However, comprehensive transcriptomic analysis related to this sex differentiation of Z. scutellata was necessary to determine effective target genes for the genetic control. We performed de novo assembly of the transcript obtained by paired-end sequencing using an Illumina HiSeq platform and let to 217,967 unigenes (i.e., unique genes) with a minimum length of 200 bp. The female produced 31, 604, 442 reads with 97.93% of Q20, 94.76% of Q30, and the male produced 130, 592, 828 reads with 97.93% of Q20 and 94.76 of Q30%. The differentially expressed genes were used to predict genetic factors associated with sex differentiation, which included Rho1, extra-macrochaetae (emc), hopscotch (hop), doublesex (dsx), sex-lethal (sxl), transformer-2 (tra-2), testis-specific serine/threonine-protein kinase (tssk1), tektin1 (tkt1) and 2 (tkt2), odorant binding proteins (OBPs), fruitless (fru), vitellogenin receptor, and hormone receptors in Z. scutellata. In addition, this transcriptome analysis provides the additional gene associated with sex determination and mating behaviors, which would be applied to develop a novel sterile insect technique against Z. scutellata.

High-efficiency removal of pyrethroids using a redesigned odorant binding protein

Pyrethroids are ubiquitously present in environmental media and threaten both the ecosystem and human health. To explore effective ways to remove pyrethroids from the environment, an odorant binding protein (OBP) with affinity for various pyrethroids was investigated. Initially, the target OBP, Spodoptera littoralis pheromone binding protein 1 (SlitPBP1), underwent redesign to enhance its affinity for pyrethroids. The modified SlitPBP1E97ND106E demonstrated a substantially increased affinity for deltamethrin (DeltaM), with a dissociation constant of 0.77 ± 0.17 μM. The affinity of SlitPBP1E97ND106E for other pyrethroids also increased to varying extents. Consequently, SlitPBP1E97ND106E displayed a markedly enhanced capability to adsorb and remove pyrethroids. When exposed to free SlitPBP1E97ND106E in solution, the reduction in DeltaM surged from 16.78 ± 0.32% to 97.51 ± 0.56%. SlitPBP1E97ND106E was immobilized by coupling the protein to Ni2+-NTA agarose resin. Liquid chromatography results attested to the superior efficacy of immobilized SlitPBP1E97ND106E in removing pyrethroids, especially DeltaM. No significant differences in pyrethroid removal were detected across various water samples. Our findings introduce a potent tool for pyrethroid removal. A wider range of OBPs can similarly be optimized and applied to remove organic pollutants, including but not limited to pesticides.

The first chromosome-level genome assembly and transcriptome sequencing provide insights into cantharidin production of the blister beetles.

Blister beetles (Coleoptera: Meloidae) produce a natural defensive toxin cantharidin (CTD), which has been used for various cancer treatments and other diseases. Currently, the lack of chromosome-level reference genomes in Meloidae limits further understanding of the mechanism of CTD biosynthesis and environmental adaptation. In this study, the chromosome-level genome assembly of Mylabris phalerata was generated based on PacBio and Hi-C sequencing. This reference genome was about 136.68 Mb in size with contig N50 of 9.17 Mb and composed of 12 chromosomes. In comparison to six other Coleoptera insects, M. phalerata exhibited multiple expanded gene families enriched in juvenile hormone (JH) biosynthetic process pathway, farnesol dehydrogenase activity, and cytochrome P450, which may be related to CTD biosynthesis. Consistently, the transcriptomic analysis suggested the "terpenoid backbone biosynthesis" pathway and "the juvenile hormone" as putative core pathways of CTD biosynthesis and presented eight up-regulated differential expression genes in male adults as candidate genes. It is possible that the restricted feeding niche and lifestyle of M. phalerata were the cause of the gene family's contraction of odorant binding proteins. The ABC transporters (ABCs) related to exporting bound toxins out of the cell and the resistance to the self-secreted toxins (e.g. CTD) were also contracted, possibly due to other self-protection strategies in M. phalerata. A foundation of understanding CTD biosynthesis and environmental adaptation of blister beetles will be established by our reference genome and discoveries.

Green synthesis and biophysical characterization of silver and palladium nanoparticles using Laureliopsis philippiana: A potent eco-friendly larvicide with negligible impact on zebrafish (Danio rerio)

Green nanoparticles represent a sustainable and eco-friendly approach to developing larvicides targeted at controlling mosquito populations. These nanoparticles are synthesized using natural, non-toxic materials and environmentally benign processes, emphasizing their potential to combat mosquito larvae while minimizing adverse effects on the ecosystem. This approach aligns with the growing need for sustainable solutions in vector control, providing an effective alternative to traditional larvicides with reduced environmental impact. The aim of the present investigation we have utilized the leaf from Laureliopsis philippiana (LP) to synthesize silver (Ag) and palladium (Pd) nanoparticles (NPs) and studied toxicity on deadly disease vectors larvae of Aedes aegypti and Anopheles stephensi. Biosynthesized Ag and Pd NPs were extensively characterized through physical and chemical methods. Gas chromatography–mass spectrometry analysis of L. philippiana assisted in identifying the secondary metabolites of the extract's total flavonoid and phenolic compounds, which are in the reactants of the bio-reduction process during the production of NPs. The in-silico analysis was conducted on mosquito sterol carrier protein-2 (1PZ4) and mosquito odorant binding protein (2L2C). The biosynthesized Ag and Pd NPs revealed the UV absorption peak at 417 and 370 nm, and X-ray diffraction results showed the crystalline structure of NPs. At the same time, in transmission electron microscopy representation, the Ag and Pd NPs were spherical, clustered, and exhibited a noticeable average size of Ag NPs: 13.25 ± 2.53 nm and Pd NPs: 4.07 ± 1.64 nm. The biogenic Ag and Pd NPs showed concentration-dependent larvicidal mortality at 12 and 24-hour exposure with LC50 values of 29.596, 17.804, and 23.915, 14.878 µg/mL on Ae. aegypti and 23.946, 13.938, 19.457, and 12.876 µg/mL against An. stephensi, correspondingly. In-silico studies proved that Laureliopsine A and isoterpinolene showed significant docking scores compared to permethrin on mosquito proteins (1PZ4 and 2L2C). Besides, the LP-Ag and Pd NPs non-toxicity analysis was carried out on zebrafish (Danio rerio) embryos at 72 h post-fertilization. Overall, the findings clearly demonstrated the green synthesized Ag and Pd NPs from L. philippiana to develop environmentally secure and safe larvicides.

Pea aphid odorant-binding protein ApisOBP6 discriminates between aphid sex pheromone components, aphid alarm pheromone and a host plant volatile

Olfactory perception of pheromones in insects involves odorant-binding proteins (OBPs), relatively small proteins (ca.110-240 amino acid residues) that can bind reversibly to behaviourally active olfactory ligands. In this study, we investigated the binding in silico and in vitro of the aphid sex pheromone components (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone and the aphid alarm pheromone (E)-β-farnesene by OBPs from the pea aphid, Acyrthosiphon pisum. Screening of protein models of ApisOBPs1-11 with the aphid sex pheromone components suggested that ApisOPB6 was a candidate. Fluorescence assays using ApisOBP6 suggested that ApisOBP6 was able to bind both sex pheromone components and discriminate from the aphid alarm pheromone and the generic plant compound (R/S)-linalool. Saturation transfer difference NMR experiments with ApisOBP6 yielded results consistent to those from the fluorescence experiments, with a clear interaction between ApisOBP6 and (4aS,7S,7aR)-nepetalactone. These results describe a novel interaction and potential function for ApisOBP6, point to pre-receptor odorant discrimination by OBPs, and provide a platform for investigating the function of other aphid olfactory proteins involved in aphid chemical ecology.

An OBP gene highly expressed in non-chemosensory tissues affects the phototaxis and reproduction of Spodoptera frugiperda.

Insect odorant binding proteins (OBPs) were initially regarded as carriers of the odorants involved in chemosensation. However, it had been observed that a growing number of OBP genes exhibited broad expression patterns beyond chemosensory tissues. Here, an OBP gene (OBP31) was found to be highly expressed in the larval ventral nerve cord, adult brain and male reproductive organ of Spodoptera frugiperda. An OBP31 knockout strain (OBP31-/- ) was generated by CRISPR/Cas9 mutagenesis. For OBP31-/- , the larvae needed longer time to pupate, but there was no difference in the pupal weight between OBP31-/- and wild type (WT). OBP31-/- larvae showed stronger phototaxis than the WT larvae, indicating the importance of OBP31 in light perception. For mating rhythm of adults, OBP31-/- moths displayed an earlier second mating peak. In the cross-pairing of OBP31-/- and WT moths, the mating duration was longer, and hatchability was lower in OBP31-/- group and OBP31+/- ♂ group than that in the WT group. These results suggested that OBP31 played a vital role in larval light perception and male reproductive process and could provide valuable insights into understanding the biological functions of OBPs that were not specific in chemosensory tissues.

Roles of insect odorant binding proteins in communication and xenobiotic adaptation.

Odorant binding proteins (OBPs) are small water-soluble proteins mainly associated with olfaction, facilitating the transport of odorant molecules to their relevant receptors in the sensillum lymph. While traditionally considered essential for olfaction, recent research has revealed that OBPs are engaged in a diverse range of physiological functions in modulating chemical communication and defense. Over the past 10 years, emerging evidence suggests that OBPs play vital roles in purifying the perireceptor space from unwanted xenobiotics including plant volatiles and pesticides, potentially facilitating xenobiotic adaptation, such as host location, adaptation, and pesticide resistance. This multifunctionality can be attributed, in part, to their structural variability and effectiveness in transporting, sequestering, and concealing numerous hydrophobic molecules. Here, we firstly overviewed the classification and structural properties of OBPs in diverse insect orders. Subsequently, we discussed the myriad of functional roles of insect OBPs in communication and their adaptation to xenobiotics. By synthesizing the current knowledge in this field, our review paper contributes to a comprehensive understanding of the significance of insect OBPs in chemical ecology, xenobiotic adaptation, paving the way for future research in this fascinating area of study.

Activity of Single Insect Olfactory Receptors Triggered by Airborne Compounds Recorded in Self-Assembled Tethered Lipid Bilayer Nanoarchitectures.

Membrane proteins are among the most difficult to study as they are embedded in the cellular membrane, a complex and fragile environment with limited experimental accessibility. To study membrane proteins outside of these environments, model systems are required that replicate the fundamental properties of the cellular membrane without its complexity. We show here a self-assembled lipid bilayer nanoarchitecture on a solid support that is stable for several days at room temperature and allows the measurement of insect olfactory receptors at the single-channel level. Using an odorant binding protein, we capture airborne ligands and transfer them to an olfactory receptor from Drosophila melanogaster (OR22a) complex embedded in the lipid membrane, reproducing the complete olfaction process in which a ligand is captured from air and transported across an aqueous reservoir by an odorant binding protein and finally triggers a ligand-gated ion channel embedded in a lipid bilayer, providing direct evidence for ligand capture and olfactory receptor triggering facilitated by odorant binding proteins. This model system presents a significantly more user-friendly and robust platform to exploit the extraordinary sensitivity of insect olfaction for biosensing. At the same time, the platform offers a new opportunity for label-free studies of the olfactory signaling pathways of insects, which still have many unanswered questions.

Computational insights of the molecular recognition between volatile molecules and odorant binding proteins from the red palm weevil Rhynchophorus ferrugineus

The red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) is one of the most harmful pests for palm trees, causing serious economic damage worldwide. The present work aims to model and study the 3D structures of highly expressed odorant binding proteins from R. ferrugineus (RferOBPs) and identify possible binding modes and ligand release mechanism by docking and molecular dynamics. Highly confident 3D structures of a total of 11 odorant binding proteins (OBPs) were obtained with AlphaFold2. All 3D RferOBPs modeled structures displayed six characteristic α-helices, except for RfeOBP7 and RfeOBP10, which had an extra terminal α-helix. Among the eleven modeled RferOBPs, RferOBP4 was highly expressed in the antennae and subsequently selected for further analyses. Molecular docking analyses demonstrated that ferruginol, α-pinene, DEET, and picaridin can favorably bind the RferOBP4 cavity with low affinity energies. Molecular dynamic simulations of RferOBP4 bound to ferruginol at different pH values showed that low pH environments dictate a structural change into an apo-state that modifies the number of tunnels where the ligand can coexist, further triggering ligand release by a pH-dependent mechanism. This is the first report concerning the modelling and study of ligand binding modes and release mechanism of R. ferrugineus OBPs. Communicated by Ramaswamy H. Sarma

Chemosensory proteins as putative semiochemical carriers in the desert isopod Hemilepistus reaumurii

The order Isopoda contains both aquatic and terrestrial species, among which Hemilepistus reaumurii, which lives in arid environments and is the most adapted to terrestrial life. Olfaction has been deeply investigated in insects while it has received very limited attention in other arthropods, particularly in terrestrial crustaceans. In insects, soluble proteins belonging to two main families, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs), are contained in the olfactory sensillar lymph and are suggested to act as carriers of hydrophobic semiochemicals to or from membrane-bound olfactory receptors. Other protein families, namely Nieman-Pick type 2 (NPC2) and Lipocalins (LCNs) have been also reported as putative odorant carriers in insects and other arthropod clades. In this study, we have sequenced and analysed the transcriptomes of antennae and of the first pair of legs of H. reaumurii focusing on soluble olfactory proteins. Interestingly, we have found 13 genes encoding CSPs, whose sequences differ from those of the other arthropod clades, including non-isopod crustaceans, for the presence of two additional cysteine residues, besides the four conserved ones. Binding assays on two of these proteins showed strong affinities for fatty acids and long-chain unsaturated esters and aldehydes, putative semiochemicals for this species.

Mammalian odorant-binding proteins are prone to form amorphous aggregates and amyloid fibrils

Odorant-binding proteins are involved in perceiving smell by capturing odorants within the protein's β-barrel. On the example of bovine odorant-binding protein (bOBP), the structural organization of such proteins and their ability to bind ligands under various conditions in vitro were examined. We found a tendency of bOBP to form oligomers and small amorphous aggregates without disturbing the integrity of protein monomers at physiological conditions. Changes in environmental parameters (increased temperature and pH) favored the formation of larger and dense supramolecular complexes that significantly reduce the binding of ligands by bOBP. The ability of bOBP to form fibrillar aggregates with the properties of amyloids, including high cytotoxicity, was revealed at sample stirring (even at physiological temperature and pH), at medium acidification or pre-solubilization with hexafluoroisopropanol. Fibrillogenesis of bOBP was initiated by the dissociation of the protein's supramolecular complexes into monomers and the destabilization of the protein's β-barrels without a significant destruction of its native β-strands.

Minus-C odorant binding protein TcasOBP7G contributes to reproduction and defense against phytochemical in the red flour beetle, Tribolium castaneum

Insect odorant binding proteins (OBPs) paly an important role in the recognition and transmission signals. One odorant binding protein gene, TcasOBP7G, was significantly up-regulated after exposure to eucalyptol. Nevertheless, the causal role of TcasOBP7G in Tribolium castaneum is still unclear. Here, TcasOBP7G was cloned and characterized from T. castaneum, aligning with the typical characteristic of Minus-C OBPs. The analysis of gene expression profile showed that TcasOBP7G is highly expressed at late larval and adult stage, with particularly high expression levels in ovary and testis of adults, suggesting that TcasOBP7G might be associated with reproduction of T. castaneum. Furthermore, RNAi of TcasOBP7G led to a decrease in spawning, while the development of gonads was normal, suggesting that TcasOBP7G may medicate the reproduction of T. castaneum. Additionally, the mRNA level of TcasOBP7G was significantly increased after exposure to eucalyptol, and knockdown of TcasOBP7G resulted in an increased susceptibility of T. castaneum to eucalyptol, indicating that TcasOBP7G is associated with the tolerance of T. castaneum to eucalyptol. Altogether, these results indicate that TcasOBP7G is required for both reproduction and defense against phytochemical in T. castaneum. This study will provide a theoretical basis for the development of novel control strategies based on the olfaction of insects.

Key Amino Acid Residues Involved in Binding Interactions between Bactrocera minax Odorant-Binding Protein 3 (BminOBP3) and Undecanol.

Insect odorant-binding proteins (OBPs) are significant in binding and transporting odorants to specific receptors. Our previous study demonstrated that BminOBP3 exhibited a strong affinity with undecanol. However, the binding mechanism between them remains unknown. Here, using homology modeling and molecular docking, we found that the C-terminus (I116-P122), especially the hydrogenbonds formed by the last three amino acid residues (V120, F121, and P122) of the C-terminus, is essential for BminOBP3's ligand binding. Mutant binding assays showed that the mutant T-OBP3 that lacks C-terminus (I116-P122) displayed a significant decrease in affinity to undecanol (Ki = 19.57 ± 0.45) compared with that of the wild-type protein BminOBP3 (Ki = 11.59 ± 0.51). In the mutant 3D2a that lacks F121 and P122 and the mutant V120A in which V120 was replaced by alanine, the bindings to undecanol were completely abolished. In conclusion, the C-terminus plays a crucial role in the binding interactions between BminOBP3 and undecanol. Based on the results, we discussed the ligand-binding process of BminOBP3.