Cytotoxin-mediated silk gland organ dysfunction diverts resources to enhance silkworm fecundity by potentiating nutrient-sensing IIS/TOR pathways.

Energy reserves, primarily stored in the insect's fat body, are essential for physiological processes such as reproduction and cocoon formation. However, whether these processes are mutually constraining is unknown. Here, we showed that cocoon-free silkworms accumulate amino acid constituents of silk proteins in the hemolymph and maintain lipid and sugar reserves in the pupal fat body by repressing the expression of sericin and fibroin genes in the middle and posterior silk glands, respectively, via butterfly pierisin-1A catalytic domain expression. This, in turn, upregulates insulin/insulin-like signaling and target of rapamycin (IIS/TOR) signaling, which enhances vitellogenesis and accelerates ovarian development, thus contributing to increased fecundity. The impacts of semi-starvation on fecundity and egg hatchability were also less pronounced in cocoon-free silkworms compared with wildtype silkworms. These data uncover the resource allocation trade-off between cocoon formation and fecundity and demonstrate that nutritional signaling plays a role in regulating silkworm reproduction.

A Bioengineering Approach for the Development of Fibroblast Growth Factor-7-Functionalized Sericin Biomaterial Applicable for the Cultivation of Keratinocytes.

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.

Development of a cypovirus protein microcrystal-encapsulated Bacillus thuringiensis UV-tolerant and mosquitocidal δ-endotoxin.

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown potential as an effective proteinaceous pesticide against mosquitoes, it has an ultraviolet (UV)-intolerant property that limits its outdoor use. Our previous research showed that protein microcrystal polyhedra from Bombyx mori cypovirus can encapsulate diverse foreign proteins and maintain long-term protein activity under hostile environmental conditions, including UV irradiation. In this study, we report the development of polyhedra encapsulating the Cry4Aa insecticidal activity domain by using a modified baculovirus expression system. We confirmed the oral intake of recombinant polyhedra introduced into the experimental environment by the larvae of a mosquito, Aedes albopictus, and delivery of encapsulated proteins into the digestive tract. The polyhedra encapsulating partial Cry4Aa showed mosquito larvicidal activity during incubation of larvae with 50% lethal-dose value of 23.717×104 cubes for 10 Aedes albopictus larvae in 1 ml water. In addition, polyhedra showed a specific property to reduce the impact of UV-C irradiation on the activity of encapsulated partial Cry4Aa, thus demonstrating the effectiveness of encapsulating Bti δ-endotoxins inside polyhedra to increase the availability of proteinaceous pesticides for outdoor use for mosquito control.

Bioengineered Silkworm for Producing Cocoons with High Fibroin Content for Regenerated Fibroin Biomaterial-Based Applications.

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1-5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.

Effects of transgenic silk materials that incorporate FGF-7 protein microcrystals on the proliferation and differentiation of human keratinocytes.

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor-7 (FGF-7) fused with the polyhedron-encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF-7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF-7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF-7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF-7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF-7-polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.

New method for immobilising diverse proteins onto cubic micro-protein polyhedrin crystals.

Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Raman spectroscopy insight into Norovirus encapsulation in Bombyx mori cypovirus cubic microcrystals.

Protein and amino acid structures of Norovirus-like particles (NoVLP) have been investigated by Raman spectroscopy before and after encapsulation into Bombyx mori cypovirus (BmCPV) cubic microcrystals, which are usually referred to as cubes or polyhedra. Two different types of tag were used in co-expression, namely VP3 and H1 tags. VP3 tag is derived from a capsid protein VP4 from BmCPV and H1 tag is N-terminal α-helix of BmCPV polyhedrin, respectively. A major capsid protein VP1 of NoVLP G11.4 was fused with H1 or VP3 tags, and then encapsulated into BmCPV polyhedra. Analyses of the spectroscopic data permitted the assignment of conformation-sensitive Raman bands to viral amino acid constituents and the observation of structural similarities or differences between differently tagged samples. Three separate Raman zones were attentioned, namely, the ring-mode structure region (1000-1500 cm-1), the CO and CC double-bond region and its surroundings (1500-1750 cm-1), and the high-frequency CH stretching region (2800-3100 cm-1). Structural fingerprints could be found in specific spectral zones for differently co-expressed samples. One clear characteristic of the H1-tagged VP1 polyhedra was the increase in tyrosine fraction, which played a critical role in binding neighboring strands through its unpaired negatively charged COO- sites. This feature could consistently be detected in different regions, but it was best represented by Raman signals associated with negatively charged COO- sites and H1 helices in the double-bond region. Such peculiar chemical features were revealed by two relatively broad bands at 1570 and 1630 cm-1, which were assigned to COO- anti-symmetric stretching and amide I in 310-helix extensions to α-helices at N-termini, respectively. These specific features did not display in the spectrum of the VP3-tagged VP1 polyhedra. Concurrently, a strong reduction of CH bond Raman signal was noticed in the high frequency stretching region of the Raman spectrum upon H1-tagged VP1 polyhedra. The Raman activity most strikingly also represented fingerprints of tagged NoVLP VP1 after its encapsulation into BmCPV polyhedra, opening thus the possibility to in situ advanced experiments in the fields of drug delivery and regenerative medicine.

Supramolecular protein cages constructed from a crystalline protein matrix.

Protein crystals are formed via ordered arrangements of proteins, which assemble to form supramolecular structures. Here, we show a method for the assembly of supramolecular protein cages within a crystalline environment. The cages are stabilized by covalent cross-linking allowing their release via dissolution of the crystal. The high stability of the desiccated protein crystals allows cages to be constructed.

Structure of in cell protein crystals containing organometallic complexes.

The molecular structures of in cell protein crystals containing organometallic Pd(allyl) complexes were determined by performing microfocus X-ray diffraction experiments. The coordination sites in a polyhedrin mutant with deletion of selected amino acid residues located at the interface of the polyhedrin trimer are dramatically altered compared to those of the wild-type composite.

Substituent Effect on the Formation of Arylindanes by Dimerization of Ferulic Acid and its Related Compounds.

Homodimerization and crossed-dimerization reactions for ferulic acid and related compounds in an acidic ethanol solution were investigated via a formal [3 + 2] cycloaddition reaction. Arylindanes, as the major products, were produced from some of the acids with hydroxy group and/or methoxy groups at the para and meta positions of the phenyl ring. The formation of arylindane required the following consecutive reactions: (I) protonation of the first produced ester to afford to a benzylic cation, (II) which reacts with another ester to afford a new benzylic cation, and (III) followed by cyclization to produce the dimer. It is suggested that the para-substituent promoted both step I and step II and the meta-substituent accelerated step III. To rationalize the selectivity of homodimerization, the stability of the benzyl cations and the specific electron distribution on the molecular surface of each ester is discussed based on the results of the molecular orbital calculations.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Crystal Engineering of Self-Assembled Porous Protein Materials in Living Cells.

Crystalline porous materials have been investigated for development of important applications in molecular storage, separations, and catalysis. The potential of protein crystals is increasing as they become better understood. Protein crystals have been regarded as porous materials because they present highly ordered 3D arrangements of protein molecules with high porosity and wide range of pore sizes. However, it remains difficult to functionalize protein crystals in living cells. Here, we report that polyhedra, a natural crystalline protein assembly of polyhedrin monomer (PhM) produced in insect cells infected by cypovirus, can be engineered to extend porous networks by deleting selected amino acid residues located on the intermolecular contact region of PhM. The adsorption rates and quantities of fluorescent dyes stored within the mutant crystals are increased relative to those of the wild-type polyhedra crystal (WTPhC) under both in vitro and in vivo conditions. These results provide a strategy for designing self-assembled protein materials with applications in molecular recognition and storage of exogenous substances in living cell as well as an entry point for development of bioorthogonal chemistry and in vivo crystal structure analysis.

Photoactivatable CO release from engineered protein crystals to modulate NF-κB activation.

Photoactivatable CO releasing protein crystals were developed by immobilization of Mn carbonyl complexes in polyhedral crystals, which are spontaneously formed in insect cells. The photoactivatable CO release from the engineered protein crystals activates nuclear factor kappa B (NF-κB) upon stimulation by visible light irradiation with suppression of cytotoxicity of the Mn complex.

Design of Enzyme-Encapsulated Protein Containers by In Vivo Crystal Engineering.

Crystalline protein assemblies of polyhedra crystal (PhC) can be utilized as solid enzyme containers for long-term storage of enzymes with retention of their enzymatic activity. The enzymes can be released from the crystals at the optimum pH for the enzymatic activity by dissolution of the crystals using in vivo crystal engineering.

Cell proliferation by silk gut incorporating FGF-2 protein microcrystals.

Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals, and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential.

Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge.

Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 × 10(7) BMP-2 polyhedra, and half were treated with sponges containing 3.6 × 10(6) BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 µg of recombinant human BMP-2 (rhBMP-2) or 1.8 × 10(7) empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development.

3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6.

Control of angiogenesis by VEGF and endostatin-encapsulated protein microcrystals and inhibition of tumor angiogenesis.

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals derived from insect cypovirus with overexpression of protein disulfide bond isomerase. VEGF microcrystals were observed to increase the phosphorylation of p42/p44 MAP kinase and to stimulate the proliferation, migration, and network and tube formation of human umbilical vein endothelial cells (HUVECs). Endostatin was also successfully encapsulated into microcrystals. Endostatin microcrystals showed antiangiogenesis activities and inhibited the migration, and network and tube formation of HUVECs. Local administration of endostatin microcrystals in mice inhibited both angiogenesis and tumor growth with clear significant differences between treatment and control groups. Endostatin microcrystals only affected angiogenesis, but had no significant effect on lymphangiogenesis compared to controls. Local therapy using endostatin microcrystals offers a potential approach to achieve sustained therapeutic release of antiangiogenic molecules for cancer treatment.