Crystal Structure of Silkworm PIWI-Clade Argonaute Siwi Bound to piRNA.

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.

The molecular basis of sugar detection by an insect taste receptor.

Animals crave sugars because of their energy potential and the pleasurable sensation of tasting sweetness. Yet all sugars are not metabolically equivalent, requiring mechanisms to detect and differentiate between chemically similar sweet substances. Insects use a family of ionotropic gustatory receptors to discriminate sugars1, each of which is selectively activated by specific sweet molecules2-6. Here, to gain insight into the molecular basis of sugar selectivity, we determined structures of Gr9, a gustatory receptor from the silkworm Bombyx mori (BmGr9), in the absence and presence of its sole activating ligand, D-fructose. These structures, along with structure-guided mutagenesis and functional assays, illustrate how D-fructose is enveloped by a ligand-binding pocket that precisely matches the overall shape and pattern of chemical groups in D-fructose. However, our computational docking and experimental binding assays revealed that other sugars also bind BmGr9, yet they are unable to activate the receptor. We determined the structure of BmGr9 in complex with one such non-activating sugar, L-sorbose. Although both sugars bind a similar position, only D-fructose is capable of engaging a bridge of two conserved aromatic residues that connects the pocket to the pore helix, inducing a conformational change that allows the ion-conducting pore to open. Thus, chemical specificity does not depend solely on the selectivity of the ligand-binding pocket, but it is an emergent property arising from a combination of receptor-ligand interactions and allosteric coupling. Our results support a model whereby coarse receptor tuning is derived from the size and chemical characteristics of the pocket, whereas fine-tuning of receptor activation is achieved through the selective engagement of an allosteric pathway that regulates ion conduction.

Study on the Structure and Properties of Silk Fibers Obtained from Factory All-Age Artificial Diets.

The traditional production mode of the sericulture industry is no longer suitable for the development requirements of modern agriculture; to facilitate the sustainable development of the sericulture industry, factory all-age artificial diet feeding came into being. Understanding the structural characteristics and properties of silk fibers obtained from factory all-age artificial diet feeding is an important prerequisite for application in the fields of textiles, clothing, biomedicine, and others. However, there have been no reports so far. In this paper, by feeding silkworms with factory all-age artificial diets (AD group) and mulberry leaves (ML group), silk fibers were obtained via two different feeding methods. The structure, mechanical properties, hygroscopic properties, and degradation properties were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Structurally, no new functional groups appeared in the AD group. Compared with the ML group, the structure of the two groups was similar, and there was no significant difference in mechanical properties and moisture absorption. The structure of degummed silk fibers is dominated by crystalline regions, but α-chymotrypsin hydrolyzes the amorphous regions of silk proteins, so that after 28 d of degradation, the weight loss of both is very small. This provides further justification for the feasibility of factory all-age artificial diets for silkworms.

Isolation and Structure Analysis of Chitin Obtained from Different Developmental Stages of the Mulberry Silkworm (Bombyx mori).

Chitin, a ubiquitous biopolymer, holds paramount scientific and economic significance. Historically, it has been primarily isolated from marine crustaceans. However, the surge in demand for chitin and the burgeoning interest in biopolymers have necessitated the exploration of alternative sources. Among these methods, the mulberry silkworm (Bombyx mori) has emerged as a particularly intriguing prospect. To isolate chitin from Bombyx mori, a chemical extraction methodology was employed. This process involved a series of meticulously orchestrated steps, including Folch extraction, demineralization, deproteinization, and decolorization. The resultant chitin was subjected to comprehensive analysis utilizing techniques such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), 13C nuclear magnetic resonance (NMR) spectroscopy, and wide-angle X-ray scattering (WAXS). The obtained results allow us to conclude that the Bombyx mori represents an attractive alternative source of α-chitin.

Revealing the synergistic effect of hydration and pulsed ultrasound on the emulsifying properties of silkworm pupa protein and its stabilized emulsion.

BACKGROUND: Silkworm (Bombyx moil L.) Pupa protein (SPP) is a high-quality insect protein and is considered a sustainable alternative source for traditional animal food protein. However, the utilization of SPP is limited because of its low solubility and emulsifying ability. In the present study, the synergistic effect of hydration and pulsed ultrasound on the physicochemical properties of SPP and SPP-stabilized Pickering emulsions was evaluated. RESULTS: Pulsed ultrasound changed the particle size of SPP and its conformation. As the pulsed ultrasound increased from 0 s to 5 s, the α-helix and SS contents of SPP decreased, whereas the ß-sheet and SH contents increased, which in turn improved its solubility and amphiphilicity. As a result, the SPP treated by a combination of 12 h of hydration and 3 s of ultrasound exhibited a contact angle of 74.95°, hydrophobicity of 904.83, EAI of 6.66 m2 g-1 and ESI of 190.69 min. Compared with the combination of 1 h of hydration and 5 s of ultrasound, the combination of 12 h of hydration and 3 s of ultrasound exerted more soluble and hydrophobic SPP, whereas the EAI and ESI of the samples were higher. Notably, the ultrasound-treated SPP can form a stable gel-like emulsion (oil fraction ranging from 70% to 80%). CONCLUSION: The combination of hydration and ultrasound can effectively improve the physicochemical characteristics of SPP as well as its emulsion stability. Sufficient hydration is a cost-effective method for facilitating the modification of proteins by ultrasound treatment. © 2024 Society of Chemical Industry.

Real-Time Monitoring of the Structural Transition of Bombyx mori Liquid Silk under Pressure by Solid-State NMR.

Silk fibroin is stored in the silk glands of Bombyx mori silkworms as a condensed aqueous solution called liquid silk. It is converted into silk fibers at the silkworm's spinnerets under mechanical forces including shear stress and pressure. However, the detailed mechanism of the structural transition of liquid silk to silk fibers under pressure is not well understood. Magic angle spinning (MAS) in solid-state nuclear magnetic resonance (NMR) can exert pressure on liquid samples in a quantitative manner. In this study, solid-state NMR was used to quantitatively analyze the impact of pressure on the structural transition of liquid silk. A combination of 13C DD-MAS and CP-MAS NMR measurements enabled the conformation and dynamics of the crystalline region of the silk fibroin (both before (Silk Ip) and after (Silk IIp) the structural transition) to be detected in real time with atomic resolution. Spectral analyses proposed that the pressure-induced structural transition from Silk Ip to Silk IIp proceeds by a two-step autocatalytic reaction mechanism. The first reaction step is a nucleation step in which Silk Ip transforms to single lamellar Silk IIp, and the second is a growth step in which the single lamellar Silk IIp acts as a catalyst that reacts with Silk Ip molecules to further form Silk IIp molecules, resulting in stacked lamellar Silk IIp. Furthermore, the rate constant in the second step shows a significant pressure dependence, with an increase in pressure accelerating the formation of large stacked lamellar Silk IIp.

Microcompartmentalization Controls Silk Feedstock Rheology.

The rheological characteristics of pre-spun native silk protein, which is stored as a viscous pulp inside the silk gland, are the key factors that determine the mechanical performance of the endpoint material: the spun silk fibers. In silkworms and arthropods, microcompartmentalization was shown to play an important regulatory role in storing and stabilizing the aggregation-prone silk and in initiating the fibrillar self-assembly process. However, our current understanding of the mechanism of stabilization of the highly unstable protein pulp in its soluble state inside the microcompartments and of the conditions required for initiating the structural transition in protein inside the microcompartments remains limited. Here, we exploited the power of droplet microfluidics to mimic the silk protein's microcompartmentalization event; we introduced changes in the chemical environment and analyzed the storage-to-spinning transition as well as the accompanying structural changes in silk fibroin protein, from its native fold into an aggregative ß-sheet-rich structure. Through a combination of experimental and computational simulations, we established the conditions under which the structural transition in microcompartmentalized silk protein is initiated, which, in turn, is reflected in changes in the silk-rich fluid behavior. Overall, our study sheds light on the role of the independent parameters of a dynamically changing chemical environment, changes in fluid viscosity, and the shear forces that act to balance silk protein self-assembly, and thus, facilitate new exploratory avenues in the field of biomaterials.

Bombyx mori Silk Fibroin and Model Peptides Incorporating Arg-Gly-Asp Motifs and Their Application in Wound Dressings.

Skin plays an important role in protecting the human body from the environment, dehydration, and infection. Burns, wounds, and disease cause the skin to lose its role, but tissue-engineered skin substitutes offer the opportunity to restore skin loss. Silk fibroin from Bombyx mori (SF) has proven to be an excellent wound dressing material. In this study, we aim to develop an excellent wound dressing material by introducing three-residue sequence Arg-Gly-Asp (RGD), which is the most well-known adhesion site of fibronectin, in the films of SF and the model peptide. Its usefulness as a wound dressing material was evaluated both in vitro and in vivo. First, we showed that the flexible structures of the RGD sequence are still maintained in SF with a rigid antiparallel ß-sheet structure using NMR in association with excellent wound dressings of SF containing RGD. Then, in in vitro experiments, two types of normal cells derived from human skin, normal human neonatal epidermal keratinocytes and normal human neonatal dermal fibroblasts, were used to evaluate the cell adhesion. On the other hand, in in vivo experiments, the study was conducted using a rat model of a whole skin layer defect wound. The results showed that the high-functionalized SF developed here has the potential to play a significant role in the field of wound dressings.

Kinetic Analysis Reveals the Role of Secondary Nucleation in Regenerated Silk Fibroin Self-Assembly.

Silk proteins obtained from the Bombyx mori silkworm have been extensively studied due to their remarkable mechanical properties. One of the major structural components of this complex material is silk fibroin, which can be isolated and processed further in vitro to form artificial functional materials. Due to the excellent biocompatibility and rich self-assembly behavior, there has been sustained interest in such materials formed through the assembly of regenerated silk fibroin feedstocks. The molecular mechanisms by which the soluble regenerated fibroin molecules self-assemble into protein nanofibrils remain, however, largely unknown. Here, we use the framework of chemical kinetics to connect macroscopic measurements of regenerated silk fibroin self-assembly to the underlying microscopic mechanisms. Our results reveal that the aggregation of regenerated silk fibroin is dominated by a nonclassical secondary nucleation processes, where the formation of new fibrils is catalyzed by the existing aggregates in an autocatalytic manner. Such secondary nucleation pathways were originally discovered in the context of polymerization of disease-associated proteins, but the present results demonstrate that this pathway can also occur in functional assembly. Furthermore, our results show that shear flow induces the formation of nuclei, which subsequently accelerate the process of aggregation through an autocatalytic amplification driven by the secondary nucleation pathway. Taken together, these results allow us to identify the parameters governing the kinetics of regenerated silk fibroin self-assembly and expand our current understanding of the spinning of bioinspired protein-based fibers, which have a wide range of applications in materials science.

Flow-Induced Protein Chain Deformation, Segmental Orientation, and Phase Separation in Native Silk Feedstock.

The ability of many arthropods to spin silk and its many uses bear testament to its importance in Nature. Despite over a century of research, however, the spinning process is still not fully understood. While it is widely accepted that flow and chain alignment may be involved, the link to protein gelation remains obscure. Using combinations of rheology, polarized light imaging, and infrared spectroscopy to probe different length scales, this work explored flow-induced gelation of native silk feedstock from Bombyx mori larvae. Protein chain deformation, orientation, and microphase separation were observed, culminating in the formation of antiparallel ß-sheet structures while the work rate during flow appeared as an important criterion. Moreover, infrared spectroscopy provided direct observations suggesting a loss of protein hydration during flow-induced gelation of fibroin in native silk feedstock, which is consistent with recently reported hypotheses.

Structural Basis of Neurohormone Perception by the Receptor Tyrosine Kinase Torso.

In insects, brain-derived Prothoracicotropic hormone (PTTH) activates the receptor tyrosine kinase (RTK) Torso to initiate metamorphosis through the release of ecdysone. We have determined the crystal structure of silkworm PTTH in complex with the ligand-binding region of Torso. Here we show that ligand-induced Torso dimerization results from the sequential and negatively cooperative formation of asymmetric heterotetramers. Mathematical modeling of receptor activation based upon our biophysical studies shows that ligand pulses are "buffered" at low receptor levels, leading to a sustained signal. By contrast, high levels of Torso develop the signal intensity and duration of a noncooperative system. We propose that this may allow Torso to coordinate widely different functions from a single ligand by tuning receptor levels. Phylogenic analysis indicates that Torso is found outside arthropods, including human parasitic roundworms. Together, our findings provide mechanistic insight into how this receptor system, with roles in embryonic and adult development, is regulated.

Structural Characteristics and Properties of Cocoon and Regenerated Silk Fibroin from Different Silkworm Strains.

Silk has attracted the attention of researchers as a biomedical and cosmetic material because of its good biocompatibility and cytocompatibility. Silk is produced from the cocoons of silkworms, which have various strains. In this study, silkworm cocoons and silk fibroins (SFs) were obtained from ten silkworm strains, and their structural characteristics and properties were examined. The morphological structure of the cocoons depended on the silkworm strains. The degumming ratio of silk ranged from 22.8% to 28% depending on the silkworm strains. The highest and lowest solution viscosities of SF were shown by 9671 and 9153, respectively, showing a 12-fold difference. The silkworm strains of 9671, KJ5, and I-NOVI showed a two-fold higher work of ruptures for the regenerated SF film than 181 and 2203, indicating that the silkworm strains considerably influence the mechanical properties of the regenerated SF film. Regardless of the silkworm strain, all silkworm cocoons showed good cell viability, making them suitable candidates for advanced functional biomaterials.

Fabrication, Structure, and Properties of Nonwoven Silk Fabrics Prepared with Different Cocoon Layers.

In this study, five different nonwoven silk fabrics were fabricated with silk fibers from different cocoon layers, and the effect of the cocoon layer on the structural characteristics and properties of the nonwoven silk fabric was examined. The diameter of the silk fiber and thickness of the nonwoven silk fabric decreased from the outer to the inner cocoon layer. More amino acids with higher hydrophilicity (serine, aspartic acid, and glutamic acid) and lower hydrophilicity (glycine and alanine) were observed in the outer layers. From the outer to the inner layer, the overall crystallinity and contact angle of the nonwoven silk fabric increased, whereas its yellowness index, moisture retention, and mechanical properties decreased. Regardless of the cocoon layer at which the fiber was sourced, the thermal stability of fibroin and sericin and good cell viability remained unchanged. The results of this study indicate that the properties of nonwoven silk fabric can be controlled by choosing silk fibers from the appropriate cocoon layers. Moreover, the findings in this study will increase the applicability of nonwoven silk fabric in the biomedical and cosmetic fields, which require specific properties for industrialization.

Simultaneous Extraction of Oil and Protein from Silkworm (Bombyx mori L.) Pupae (Lueng Parroj var.) and Their In Vitro Skin Moisturization.

Oil and protein from silkworm (Bombyx mori var. Leung Pairoj) pupae, by-product from sericulture, were extracted and evaluated for their potential uses as skin biomoisturizer. The silkworm pupae (SWP) oil and protein were simultaneously extracted by using three-phase partitioning (TPP) method and determined for their physicochemical properties including fatty acid and amino acid content, respectively. The highest yields of oil and protein at 8.24 ± 0.21% and 8.41 ± 0.26% w/w, respectively were obtained from 18 h extraction. Fatty acid analysis of SWP oil was rich in linolenic acid (37.81 ± 0.34%), oleic acid (28.97 ± 0.13%), palmitic acid (21.27 ± 0.05%), stearic acid (6.60 ± 0.09%) and linoleic acid (4.73 ± 0.21%). The clear yellow SWP oil possessed saponification value of 191.51 mg/g, iodine value of 119.37 g I2/g and peroxide value of 2.00 mg equivalent O2/kg. The SWP protein composed of 17 amino acids which aspartic acid, glutamic acid, glycine and serine were the major residues. SDS-PAGE analysis revealed that the SWP protein consisted of distinct protein at around 51, 70, 175 and over 175 kDa. Cytotoxicity of the SWP oil and protein was evaluated by using MTT assay and they showed low cytotoxicity toward keratinocyte cell (HaCat cell line). The SWP oil provided moisturizing effect on pig skin comparable to olive oil, while 1% and 2% of SWP protein showed higher moisturizing efficacy than 3% hydrolyzed collagen. The study indicated that the SWP oil and protein could be potential biomoisturizers for cosmetic products.

The Size Effect of Silver Nanoparticles on Reinforcing the Mechanical Properties of Regenerated Fibers.

Regenerated silk fibroin (RSF), made from discarded silk cocoons, can be processed into regenerated silk fibers by a simple, inexpensive, and environmentally friendly wet-spinning process. However, the breaking strength and toughness of most RSF fibers are lower than those of natural silk. In this study, Ag nanoparticles (NPs) of different sizes were introduced into RSF to form RSF/AgNPs hybrid fibers by wet spinning. The effects of AgNPs of different sizes on the mechanical properties and structure of the hybrid fibers were investigated. The results demonstrated that the mechanical properties of hybrid fibers were significantly improved, especially the breaking strain, after the addition of four different sizes of AgNPs. With the reduction in AgNPs size (2-60 nm), the breaking strength and breaking strain of hybrid fibers tended to increase. The results showed that the hybrid fibers containing 2 nm AgNPs were remarkable, with excellent mechanical properties and toughness, and the breaking strain reached 138.27%, which was far greater than blank RSF fibers (15.02%) and even natural silk (about 21%). The S-FTIR and WAXD showed that, compared with the larger AgNPs, the smaller AgNPs contributed more to the formation of silk fibroin ß-sheet and crystallinity, and reduced the ß-crystallite size. This study is helpful to understand the relationship between the size of nanoparticles and the mechanical properties of hybrid fibers.

Vibrational Study on the Structure, Bioactivity, and Silver Adsorption of Silk Fibroin Fibers Grafted with Methacrylonitrile.

Natural fibers have received increasing attention as starting materials for innovative applications in many research fields, from biomedicine to engineering. Bombyx mori silk fibroin has become a material of choice in the development of many biomedical devices. Grafting represents a good strategy to improve the material properties according to the desired function. In the present study, Bombyx mori silk fibroin fibers were grafted with methacrylonitrile (MAN) with different weight gains. The potential interest in biomedical applications of MAN functionalization relies on the presence of the nitrile group, which is an acceptor of H bonds and can bind metals. IR and Raman spectroscopy were used to characterize the grafted samples and the possible structural changes induced by grafting. Afterward, the same techniques were used to study the bioactivity (i.e., the calcium phosphate nucleation ability) of MAN-grafted silk fibroins after ageing in simulated body fluid (SBF) for possible application in bone tissue engineering, and their interaction with Ag+ ions, for the development of biomaterials with enhanced anti-microbial properties. MAN was found to efficiently polymerize on silk fibroin through polar amino acids (i.e., serine and tryptophan), inducing an enrichment in silk fibroin-ordered domains. IR spectroscopy allowed us to detect the nucleation of a thin calcium phosphate layer and the uptake of Ag+ ions through the nitrile group, which may foster the application of these grafted materials in biomedical applications.

The Study of Alanine Transaminase Activity in Tissues of Silkworm (Bombyx mori) via Direct Analysis in Real-Time (DART) Mass Spectrometry.

Alanine transaminase (ALT) is an important amino acid-metabolizing enzyme in silkworm Bombyx mori L., and is mainly involved in transferring glutamate to alanine (serving as an essential precursor in silk protein synthesis) through transamination. Therefore, it is generally believed that silk protein synthesis in the silk gland and the cocoon quantity increase with the increase in ALT activity to a certain extent. Here, a novel analytical method was developed to determine the ALT activity in several key tissues of Bombyx mori L. including the posterior silk gland, midgut, fat body, middle silk gland, trachea and hemolymph, by combining the direct-analysis-in-real-time (DART) ion source with a triple-quadrupole mass spectrometer. In addition, a traditional ALT activity assay, the Reitman-Frankel method, was also used to measure ALT activity for comparison. The ALT activity results obtained via the DART-MS method are in good agreement with those obtained via the Reitman-Frankel method. However, the present DART-MS method provides a more convenient, rapid and environmentally friendly quantitative method for ALT measurement. Especially, this method can also monitor ALT activity in different tissues of Bombyx mori L. in real time.

Amino Acid Substitutions at P1 Position Change the Inhibitory Activity and Specificity of Protease Inhibitors BmSPI38 and BmSPI39 from Bombyx mori.

It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid-base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors.

Cold-stored mulberry leaves affect antioxidant system and silk proteins of silkworm (Bombyx mori) larva.

BACKGROUND: Cold storage has been widely used to maintain the quality of vegetables, but whether eating cold-stored vegetables affects health remains unknown. RESULTS: This study used silkworms as an animal model to evaluate the effects of nutrient changes in cold-stored mulberry leaves (CSML) on health. Compared with fresh mulberry leaves (FML), CSML contained lower vitamin C, soluble sugars and proteins, and higher H2 O2 , suggesting decreased antioxidant ability and nutrition. The CSML did not obviously affect larval survival rate, body weight or dry matter rate, cocoon shape, weight and size, or final rates of cluster and cocooning relative to the FML, suggesting CSML did not alter overall growth and development. However, the CSML increased the initial rates of cluster and cocooning and upregulated BmRpd3, suggesting CSML shortened larval lifespan and enhanced senescence. CSML upregulated BmNOX4, downregulated BmCAT, BmSOD and BmGSH-Px and increased H2 O2 in silkworms, suggesting CSML caused oxidative stress. CSML upregulated ecdysone biosynthesis and inactivation genes and elevated ecdysone concentration in silkworms, suggesting that CSML affected hormone homeostasis. CSML upregulated apoptosis-related genes, downregulated sericin and silk fibroin genes and decreased sericin content rate in silkworms, suggesting oxidative stress and protein deficiency. CONCLUSION: Cold storage reduced nutrition and antioxidant capability of mulberry leaves. CSML did not influence growth and development of silkworm larva, but affected health by causing oxidative stress and reducing protein synthesis. The findings show that the ingredient changes in CSML had negative effects on health of silkworms. © 2023 Society of Chemical Industry.

An ionic lock and a hydrophobic zipper mediate the coupling between an insect pheromone receptor BmOR3 and downstream effectors.

Pheromone receptors (PRs) recognize specific pheromone compounds to guide the behavioral outputs of insects, which are the most diverse group of animals on earth. The activation of PRs is known to couple to the calcium permeability of their coreceptor (Orco) or putatively with G proteins; however, the underlying mechanisms of this process are not yet fully understood. Moreover, whether this transverse seven transmembrane domain (7TM)-containing receptor is able to couple to arrestin, a common effector for many conventional 7TM receptors, is unknown. Herein, using the PR BmOR3 from the silk moth Bombyx mori and its coreceptor BmOrco as a template, we revealed that an agonist-induced conformational change of BmOR3 was transmitted to BmOrco through transmembrane segment 7 from both receptors, resulting in the activation of BmOrco. Key interactions, including an ionic lock and a hydrophobic zipper, are essential in mediating the functional coupling between BmOR3 and BmOrco. BmOR3 also selectively coupled with Gi proteins, which was dispensable for BmOrco coupling. Moreover, we demonstrated that trans-7TM BmOR3 recruited arrestin in an agonist-dependent manner, which indicates an important role for BmOR3-BmOrco complex formation in ionotropic functions. Collectively, our study identified the coupling of G protein and arrestin to a prototype trans-7TM PR, BmOR3, and provided important mechanistic insights into the coupling of active PRs to their downstream effectors, including coreceptors, G proteins, and arrestin.