Molecular mechanism analysis of apoptosis induced by silk fibroin peptides.

Silk fibroin derived from silkworm cocoons exhibits excellent mechanical properties, good biocompatibility, and low immunogenicity. Previous studies showed that silk fibroin had an inhibitory effect on cells, suppressing proliferation and inducing apoptosis. However, the source of the toxicity and the mechanism of apoptosis induction are still unclear. In this study, we hypothesized that the toxicity of silk fibroin might originate from the crystalline region of the heavy chain of silk fibroin. We then verified the hypothesis and the specific induction mechanism. A target peptide segment was obtained from α-chymotrypsin. The potentially toxic mixture of silk fibroin peptides (SFPs) was separated by ion exchange, and the toxicity was tested by an MTT assay. The results showed that SFPs obtained after 4 h of enzymatic hydrolysis had significant cytotoxicity, and SFPs with isoelectric points of 4.0-6.8 (SFPα II) had a significant inhibitory effect on cell growth. LC-MS/MS analysis showed that SFPα II contained a large number of glycine-rich and alanine-rich repetitive sequence polypeptides from the heavy-chain crystallization region. A series of experiments showed that SFPα II mediated cell death through the apoptotic pathway by decreasing the expression of Bcl-2 protein and increasing the expression of Bax protein. SFPα II mainly affected the p53 pathway and the AMPK signaling pathway in HepG2 cells. SFPα II may indirectly increase the expression of Cers2 by inhibiting the phosphorylation of EGFR, which activated apoptotic signaling in the cellular mitochondrial pathway and inhibited the Akt/NF-κB pathway by increasing the expression of PPP2R2A.

Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

Sustainable Bombyx mori's silk fibroin for biomedical applications as a molecular biotechnology challenge: A review.

Silk is a natural engineering material with a unique set of properties. The major constituent of silk is fibroin, a protein widely used in the biomedical field because of its mechanical strength, toughness and elasticity, as well as its biocompatibility and biodegradability. The domestication of silkworms allows large amounts of fibroin to be extracted inexpensively from silk cocoons. However, the industrial extraction process has drawbacks in terms of sustainability and the quality of the final medical product. The heterologous production of fibroin using recombinant DNA technology is a promising approach to address these issues, but the production of such recombinant proteins is challenging and further optimization is required due to the large size and repetitive structure of fibroin's DNA and amino acid sequence. In this review, we describe the structure-function relationship of fibroin, the current extraction process, and some insights into the sustainability of silk production for biomedical applications. We focus on recent advances in molecular biotechnology underpinning the production of recombinant fibroin, working toward a standardized, successful and sustainable process.

Characterization and promotion of endothelialization of Bombyx mori silk fibroin functionalized with REDV peptide.

In the development of small-diameter vascular grafts, it is crucial to achieve early-stage endothelialization to prevent thrombus formation and intimal hyperplasia. Silk fibroin (SF) from Bombyx mori is commonly used for such grafts. However, there is a need to expedite endothelialization post-implantation. In this study, we functionalized SF with Arg-Glu-Asp-Val (REDV) (SF + REDV) using cyanuric chloride to enhance endothelialization. The immobilization of REDV onto SF was confirmed and the amount of immobilized REDV could be calculated by 1H NMR. Furthermore, the conformational changes in Tyr, Ser, and Ala residues in [3-13C]Tyr- and [3-13C]Ser-SF due to REDV immobilization were monitored using 13C solid-state NMR. The REDV immobilized onto the SF film was found to be exposed on the film's surface, as confirmed by biotin-avidin system. Cell culture experiments, including adhesiveness, proliferation, and extensibility, were conducted using normal human umbilical vein endothelial cells (HUVEC) and normal human aortic smooth muscle cells (HAoSMC) on both SF and SF + REDV films to evaluate the impact of REDV on endothelialization. The results indicated a trend towards promoting HUVEC proliferation while inhibiting HAoSMC proliferation. Therefore, these findings suggest that SF + REDV may be more suitable than SF alone for coating small-diameter SF knitted tubes made of SF threads.

The ratio of fibroin to sericin in the middle silk gland of Bombyx mori and its correlation with the extensional behavior of the silk dope.

Understanding how native silk spinning occurs is crucial for designing artificial spinning systems. One often overlooked factor in Bombyx mori is the secretion of sericin proteins. Herein, we investigate the variation in amino acid content at different locations in the middle silk gland (MSG) of B. mori. This variation corresponds to an increase in sericin content when moving towards the anterior region of the MSG, while the posterior region predominantly contains fibroin. We estimate the mass ratio of sericin to fibroin to be ~25/75 wt% in the anterior MSG, depending on the fitting method. Then, we demonstrate that the improvement in the extensional behavior of the silk dope in the MSG correlates with the increase in sericin content. The addition of sericin may decrease the viscosity of the silk dope, a factor associated with an increase in the spinnability of silk. We further discuss whether this effect could also result from other known physicochemical changes within the MSG.

Decoding silkworm spinning programmed by pH and metal ions.

Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo. Here, we found that amphipol and digitonin stabilized the structure of natural silk fibroin (NSF) by a large-scale screening in vitro, and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen. Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm, rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules. Metal ions were required for NSF nanofibril formation. The successive pH decrease from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity, thus inducing the sol-gelation transition of NSF nanofibrils. NSF nanofibrils were randomly dispersed from PSG to ASG-1, and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning. Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and pH gradient, which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.

Overexpression of bond-forming active protein for efficient production of silk with structural changes and properties enhanced in silkworm.

Silkworm silk exhibits excellent mechanical properties, biocompatibility, and has potential applications in the biomedical sector. This study focused on enhancing the mechanical properties of Bombyx mori silk by overexpressing three bond-forming active proteins (BFAPs): AFP, HSP, and CRP in the silk glands of silkworms. Rheological tests confirmed increased viscoelasticity in the liquid fibroin stock solution of transgenic silkworms, and dynamic mechanical thermal analysis (DMTA) indicated that all three BFAPs participated in the interactions between fibroin molecular networks in transgenic silk. The mechanical property assay indicated that all three BFAPs improved the mechanical characteristics of transgenic silk, with AFP and HSP having the most significant effects. A synchrotron radiation Fourier transform infrared spectroscopy assay showed that all three BFAPs increased the ß-sheet content of transgenic silk. Synchrotron radiation wide-angle X-ray diffraction assay showed that all three BFAPs changed the crystallinity, crystal size, and orientation factor of the silk. AFP and HSP significantly improved the mechanical attributes of transgenic silk through increased crystallinity, refined crystal size, and a slight decrease in orientation. This study opens new possibilities for modifying silk and other fiber materials.

Functional modification of silk fibroin from silkworms and its application to medical biomaterials: A review.

Silk fibroin (SF) from the silkworm Bombyx mori is a fibrous protein identified as a widely suitable biomaterial due to its biocompatibility, tunable degradation, and mechanical strength. Various modifications of SF protein can give SF fibers new properties and functions, broadening their applications in textile and biomedical industries. A diverse array of functional modifications on various forms of SF has been reported. In order to provide researchers with a more systematic understanding of the types of functional modifications of SF protein, as well as the corresponding applications, we comprehensively review the different types of functional modifications, including transgenic modification, modifications with chemical groups or biologically active substance, cross-linking and copolymerization without chemical reactions, their specific modification methods and applications. Furthermore, recent applications of SF in various medical biomaterials are briefly discussed.

Super silkworm cocoons constructed by multi-silkworm larvae: Promising composites with dense structures and excellent mechanical properties.

A normal silkworm cocoon (NSC) with a unique nonwoven structure is usually spun by a single silkworm larva. Notably, there is a special Bombyx mori genetic resource that many (three or more) mature larvae tend to collectively spin into one cocoon, which was named "multi-silkworm cocoon" ("MSC"). However, the MSCs display loose structure and poor mechanical properties which limits their further application. In this study, a series of hybrid silkworm cocoons (HMSCs) are obtained by hybridizing "MSC" with a selected commercial silkworm strain successfully. The morphology, microstructures, and mechanical properties of cocoons constructed by one to three silkworm larvae were characterized and compared. The results indicated that about 48.3 % of silkworm larvae could create double and triple cocoons in the F1 generation of the silkworm hybrid, displaying robust fiber networks and dense structures. The mechanical characteristics of the HMSCs, including the tensile, peeling, compression, and needle penetration resistance properties, exceeded those of MSCs, showing significant application potential for high-performance bio-composites. This study provides a practical approach for obtaining silkworm cocoons with controllable structures and mechanical properties to develop and fabricate natural composite and biomimetic materials.

An atomistic scale simulation study of structural properties in the silk-fibrohexamerin complex.

The use of Bombyx mori silk fibroin in composite materials has been extensively explored in many studies, owing to its remarkable mechanical properties. Recently, the N-glycan-engineered P25 protein was utilized to improve the mechanical properties of silk. However, the mechanism by which N-glycan-engineered P25 protein enhances the mechanical properties of silk remains unclear. This study analyzed the interaction between the P25 protein and silkworm silk using quantum mechanics/molecular mechanics multiscale simulations and discovered stronger hydrogen bonding between the amorphous domain and the P25 protein. The results confirmed that glycoengineering of the mannose molecule in N-glycan in orders of three, five, and seven increased the hydrogen bonding of the amorphous structures. However, P25 has fewer binding interactions with the crystalline domain. Silk amino acids and mannose molecules were analyzed using QM simulations, and hydroxyl and charged amino acids in the amorphous domains were found to have relatively higher reactivity with mannose molecules in N-glycans than basic and aliphatic amino acids in the crystalline domain. This study demonstrates how the N-glycan-engineered P25 protein can improve the mechanical properties of silk fibroin and identifies a key factor for N-glycan-engineered proteins.

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.

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.

[Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity].

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His6-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His6-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

Fine mapping of Green a, Ga, on chromosome 27 in Bombyx mori.

Some strains of silkworms produce green cocoons of varying intensities. This results from quantitative and qualitative differences in flavonoid pigments, which are influenced by the environment and genetic background. We discovered that the appearance of a faint green cocoon is regulated by a gene (G27) located on chromosome 27. Through mating experiments, we found that G27 is identical to an essential flavonoid cocoon gene, Ga. This locus has not been previously described. Furthermore, we narrowed down the Ga region to 438 kbp using molecular markers. Within this region, several predicted genes for sugar transporters form a cluster structure, suggesting that Ga is among them.

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.

Silk Acid as an Implantable Biomaterial for Tissue Regeneration.

Silk fibroin derived from the domesticated silkworm Bombyx mori is a protein-based biopolymer with low immunogenicity, intrinsic biodegradability, and tunable mechanical properties, showing great potential in biomedical applications. Using chemical modification to alter the primary structure of silk fibroin enables the expanded generation of new silk-based biomaterials. Inspired by the molecular structure of hyaluronic acid, which is enriched in carboxyl groups, an efficient method with scaling-up potential to achieve controlled carboxylation of silk fibroin to prepare silk acid (SA) is reported, and the biological properties of SA are further studied. The SA materials show tunable hydrophilicity and enzymatic degradation properties at different carboxylation degrees (CDs). Subcutaneous implantation in mice for up to 1 month reveals that the SA materials with a high CD present enhanced degradation while causing a mild foreign-body response, including a low inflammatory response and reduced fibrotic encapsulation. Immunofluorescence analysis further indicates that the SA materials show pro-angiogenesis properties and promote M2-type macrophage polarization to facilitate tissue regeneration. This implies great promise for SA materials as a new implantable biomaterial for tissue regeneration.

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.

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.

A review on the structure of Bombyx mori silk fibroin fiber studied using solid-state NMR: An antipolar lamella with an 8-residue repeat.

Silk fibroin (SF) fiber from the silkworm Bombyx mori in the Silk II form has been used as an excellent textile fiber for over 5000 years. Recently it has been developed for a range of biomedical applications. Further expansion of these uses builds on the excellent mechanical strength of SF fiber, which derives from its structure. This relationship between strength and SF structure has been studied for over 50 years, but it is still not well understood. In this review, we report the use of solid-state NMR to study stable-isotope labeled SF fiber and stable-isotope labeled peptides including (Ala-Gly)15 and (Ala-Gly-Ser-Gly-Ala-Gly)5 as models of the crystalline fraction. We show that the crystalline fraction is a lamellar structure with a repetitive folding using ß-turns every eighth amino acid, and that the sidechains adopt an antipolar arrangement rather than the more well-known polar structure described by Marsh, Corey and Pauling (that is, the Ala methyls in each layer point in opposite directions in alternate strands). The amino acids Ser, Tyr and Val are the next most common in B. mori SF after Gly and Ala, and occur in the crystalline and semi-crystalline regions, probably defining the edges of the crystalline region. Thus, we now have an understanding of the main features of Silk II but there is still a long way to go.

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.