Alkyl anchor-modified artificial viral capsid budding outside-to-inside and inside-to-outside giant vesicles.

The budding of human immunodeficiency virus from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Herein, we report the construction of an artificial viral capsid budding system from a lipid bilayer of GUV. The C-terminus of the ß-annulus peptide was modified using an octyl chain as an alkyl anchor via a disulfide bond. The self-assembly of the ß-annulus peptide with an octyl chain formed an artificial viral capsid aggregate. The fluorescence imaging and transmission electron microscopy observations revealed that the addition of the tetramethylrhodamine (TMR)-labeled octyl chain-bearing ß-annulus peptide to the outer aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle outside-to-inside the mother GUV. Conversely, the encapsulation of the TMR-labeled octyl chain-bearing ß-annulus peptide in the inner aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle inside-to-outside the mother GUV. Contrarily, the addition of the TMR-labeled ß-annulus peptide to GUV barely induced budding. It was demonstrated that the higher the membrane fluidity of GUV, the more likely budding would be induced by the addition of the alkyl anchor-modified artificial viral capsid. The simple virus-mimicking material developed in this study, which buds off through membrane anchoring, can provide physicochemical insights into the mechanisms of natural viral budding from cells.

Construction of an artificial viral budding system of GUVs using only synthesized molecules remains challenging. This study firstly demonstrates that budding outside-to-inside and inside-to-outside GUVs are induced by addition of alkyl anchor-modified artificial viral capsid.

Antigen/Adjuvant-Displaying Enveloped Viral Replica as a Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidate.

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled ß-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

Reversible Photocontrol of Microtubule Stability by Spiropyran-Conjugated Tau-Derived Peptides.

Spatiotemporal modulation of microtubules by light has become an important aspect of the biological and nanotechnological applications of microtubules. We previously developed a Tau-derived peptide as a binding unit to the inside of microtubules. Here, we conjugated the Tau-derived peptide to spiropyran, which is reversibly converted to merocyanine by light, as a reversible photocontrol system to stabilize microtubules. Among the synthesized peptides with spiropyran/merocyanine at different positions, several peptides were bound to the inside of microtubules and stabilized the structures of microtubules. The peptide with spiropyran at the N-terminus induced polymerization and stabilization of microtubules, whereas the same peptide with the merocyanine form did not exert these effects. Reversible formation of microtubules/tubulin aggregates was achieved using the peptide with spiropyran conjugated at the N-terminus and irradiation with UV and visible light. Spiropyran-conjugated Tau-derived peptides would be useful for spatiotemporal modulation of microtubule stability through reversible photocontrol of binding.

Anticancer Activity of Reconstituted Ribonuclease S-Decorated Artificial Viral Capsid.

Ribonuclease S (RNase S) is an enzyme that exhibits anticancer activity by degrading RNAs within cancer cells; however, the cellular uptake efficiency is low due to its small molecular size. Here we generated RNase S-decorated artificial viral capsids with a size of 70-170 nm by self-assembly of the ß-annulus-S-peptide followed by reconstitution with S-protein at neutral pH. The RNase S-decorated artificial viral capsids are efficiently taken up by HepG2 cells and exhibit higher RNA degradation activity in cells compared with RNase S alone. Cell viability assays revealed that RNase S-decorated capsids have high anticancer activity comparable to that of standard anticancer drugs.

Mechanistic Studies for the Rational Design of Multivalent Glycodendrimers.

We have synthesized B-antigen-displaying dendrimers (16-mers) with different sizes and evaluated their affinity to their IgM antibody in order to investigate which design features lead to effective multivalency. Unexpectedly, the smallest dendrimer, which cannot chelate the multiple binding sites of IgM, clearly exhibited multivalency, together with an affinity similar to or higher than those of the larger dendrimers. These results indicate that the statistical rebinding model, which involves the rapid exchange of clustered glycans, significantly contributes to the multivalency of glycodendrimers. Namely, in the design of glycodendrimers, high-density glycan presentation to enhance statistical rebinding should be considered in addition to the ability to chelate multiple binding sites. This notion stands in contrast to the currently prevailing scientific consensus, which prioritizes the chelation model. This study thus provides new and important guidelines for molecular design of glycodendrimers.

Magnetic Force-Induced Alignment of Microtubules by Encapsulation of CoPt Nanoparticles Using a Tau-Derived Peptide.

Construction of magnetotactic materials is a significant challenge in nanotechnology applications such as nanodevices and nanotransportation. Artificial magnetotactic materials can be designed from magnetotactic bacteria because these bacteria use magnetic nanoparticles for aligning with and moving within magnetic fields. Microtubules are attractive scaffolds to construct magnetotactic materials because of their intrinsic motility. Nonetheless, it is challenging to magnetically control their orientation while retaining their motility by conjugating magnetic nanoparticles on their outer surface. Here we solve the issue by encapsulating magnetic cobalt-platinum nanoparticles inside microtubules using our developed Tau-derived peptide that binds to their internal pockets. The in situ growth of cobalt-platinum nanoparticles resulted in the formation of a linear-chain assembly of nanoparticles inside the microtubules. The magnetic microtubules significantly aligned with a high order parameter (0.71) along the weak magnetic field (0.37 T) and showed increased motility. This work provides a new concept for designing magnetotactic materials.

Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of ß-Annulus Peptide into Artificial Viral Capsid.

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer ß-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of ß-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified ß-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (Kd) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.

Construction of Ribonuclease-Decorated Artificial Virus-like Capsid by Peptide Self-assembly.

Artificial virus-like capsids decorated with ribonuclease S (RNase S) on their exterior were constructed by the self-assembly of ß-annulus-S-peptide and the interaction between S-peptide moiety and S-protein. The ß-annulus-S-peptide was synthesized by native chemical ligation of ß-annulus-SBz peptide with Cys-containing S-peptide that self-assembled into artificial virus-like capsids of approximately 47 nm in size. Reconstruction of RNase S on the artificial virus-like capsids afforded spherical assembly attached small spheres on the surface, which retained ribonuclease activity.

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates.

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.

Peptide Nanomaterials Designed from Natural Supramolecular Systems.

Natural supramolecular assemblies exhibit unique structural and functional properties that have been optimized over the course of evolution. Inspired by these natural systems, various bio-nanomaterials have been developed using peptides, proteins, and nucleic acids as components. Peptides are attractive building blocks because they enable the important domains of natural protein assemblies to be isolated and optimized while retaining the original structures and functions. Furthermore, the peptide subunits can be conjugated with exogenous molecules such as peptides, proteins, nucleic acids, and metal nanoparticles to generate advanced functions. In this personal account, we summarize recent progress in the construction of peptide-based nanomaterial designed from natural supramolecular systems, including (1) artificial viral capsids, (2) self-assembled nanofibers, and (3) protein-binding motifs. The peptides inspired by nature should provide new design principles for bio-nanomaterials.

[Exhaustive analysis of genetic mutations associated with protein S deficiency utilizing next-generation sequencing analysis].

Protein S (PS) gene (PROS1) is found on chromosome 3 (3q11.1). To date, the reported detection rate of causative gene mutations in patients suspected of PS deficiency is only approximately 50%. To improve the detection rate of causative mutations, an exhaustive analysis of PROS1 was attempted using the next-generation sequencing method (NGS) to analyze the entire nucleotide sequence of PROS1 without analyzing those affected by pseudogenes. A total of 10 different mutations (three males and six females (52.9%) out of 17 patients (3 males and 14 females) with clinical PS deficiency were identified in this study. Remarkable improvements in the detection rate of causative mutations could not be obtained even with NGS analysis. These results suggested that the rate of diagnosis did not improve even after performing an exhaustive genetic analysis in patients clinically diagnosed with low PS antigen level and/or low PS activity. Although no reports were found on the gender gap in the rate of gene diagnosis for PS deficiency, the fluctuation of estrogen levels especially in women might cause a lower rate of diagnosis.

Molecular Encapsulation Inside Microtubules Based on Tau-Derived Peptides.

Microtubules are cytoskeletal filaments that serve as attractive scaffolds for developing nanomaterials and nanodevices because of their unique structural properties. The functionalization of the outer surface of microtubules has been established for this purpose. However, no attempts have been made to encapsulate molecules inside microtubules with 15 nm inner diameter. The encapsulation of various molecular cargos inside microtubules constitutes a new concept for nanodevice and nanocarrier applications of microtubules. Here, we developed peptide motifs for binding to the inner surface of microtubules, based on a repeat domain of the microtubule-associated protein Tau. One of the four Tau-derived peptides, 2N , binds to a taxol binding pocket of ß-tubulin located inside microtubules by preincubation with tubulin dimer and subsequent polymerization of the peptide-tubulin complex. By conjugation of 2N to gold nanoparticles, encapsulation of gold nanoparticles inside microtubules was achieved. The methodology for molecular encapsulation inside microtubules by the Tau-derived peptide is expected to advance the development of microtubule-based nanomaterials and nanodevices.

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified ß-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a ß-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated ß-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

Enveloped Viral Replica Equipped with Spike Protein Derived from SARS-CoV-2.

Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from ß-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.

Photoresponsive peptide materials: Spatiotemporal control of self-assembly and biological functions.

Peptides work as both functional molecules to modulate various biological phenomena and self-assembling artificial materials. The introduction of photoresponsive units to peptides allows the spatiotemporal remote control of their structure and function upon light irradiation. This article overviews the photoresponsive peptide design, interaction with biomolecules, and applications in self-assembling materials over the last 30 years. Peptides modified with photochromic (photoisomerizable) molecules, such as azobenzene and spiropyran, reversibly photo-controlled the binding to biomolecules and nanostructure formation through self-assembly. Photocleavable molecular units irreversibly control the functions of peptides through cleavage of the main chain and deprotection by light. Photocrosslinking between peptides or between peptides and other biomolecules enhances the structural stability of peptide assemblies and complexes. These photoresponsive peptides spatiotemporally controlled the formation and dissociation of peptide assemblies, gene expressions, protein-drug interactions, protein-protein interactions, liposome deformation and motility, cytoskeleton structure and stability, and cell functions by appropriate light irradiation. These molecular systems can be applied to photo-control biological functions, molecular robots, artificial cells, and next-generation smart drug delivery materials.

Dramatic morphological changes in liposomes induced by peptide nanofibers reversibly polymerized and depolymerized by the photoisomerization of spiropyran.

Cytoskeletons such as microtubules and actin filaments are natural protein assemblies, which dynamically control cellular morphology by reversible polymerization/depolymerization. Recently, the control of polymerization/depolymerization of fibrous protein/peptide assemblies by external stimuli has attracted significant attention. However, as far as we know, the creation of an "artificial cytoskeleton" that reversibly controls the polymerization/depolymerization of peptide nanofiber in giant unilamellar vesicles (GUVs) has not been reported. Here, we developed peptide nanofiber self-assembled from spiropyran (SP)-modified ß-sheet-forming peptides, which can be reversibly polymerized/depolymerized by light. The reversible photoisomerization of the SP-modified peptide (FKFECSPKFE) to the merocyanine-peptide (FKFECMCKFE) by ultraviolet (UV) and visible light irradiation was confirmed by UV-visible spectroscopy. Confocal laser scanning microscopy with thioflavin T staining and transmission electron microscopy of the peptides showed that the SP-peptide formed ß-sheet nanofibers, whereas the photoisomerization to the merocyanine-peptide almost completely dissociated the nanofibers. The merocyanine peptide was encapsulated in spherical GUVs comprising of phospholipids as artificial cell models. Interestingly, the morphology of GUV encapsulating the merocyanine-peptide dramatically changed into worm-like vesicles by the photoisomerization to the SP-modified peptide, and then reversibly changed into spherical GUV by the photoisomerization to the MC-modified peptide. These dynamic morphological changes in GUVs by light can be applied as components of a molecular robot with artificially controlled cellular functions.

A supramolecular system mimicking the infection process of an enveloped virus through membrane fusion.

Membrane fusion is an essential step for the entry of enveloped viruses, such as human immunodeficiency virus and influenza virus, into the host cell, often triggered by the binding of membrane proteins on the viral envelope to host cell membrane. Recently, external stimuli was shown to trigger membrane fusion in an artificial system. Direct observation of artificial membrane fusion using a giant unilamellar vesicle (GUV), which is similar in size to a cell, is useful as a biological model system. However, there are no model systems for studying membrane fusion of enveloped viruses with host cells. Here, we report a supramolecular model system for viral entry into a GUV or cell through membrane fusion. The system was constructed by complexing a cationic lipid bilayer on an anionic artificial viral capsid, self-assembled from viral ß-annulus peptides. We demonstrate that the cationic enveloped artificial viral capsid electrostatically interacts with the anionic GUV or cell, and the capsid enters the GUV or cell through membrane fusion. The model system established in this study will be important for analyzing membrane fusion during infection of a natural virus.

Binding of Tau-derived peptide-fused GFP to plant microtubules in Arabidopsis thaliana.

Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro. However, there was no investigation of TPs on microtubules in planta. Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules.

An artificial viral capsid decorated with a DNA aptamer internalizing into lymphoma cells.

Tumor-specific drug-delivering nanocarriers could be a promising modality for next-generation tumor therapy. Here we developed a Burkitt lymphoma-specific DNA aptamer-labeled nanocarrier using the ß-Annulus peptide, which forms a spherical nanoassembly called artificial viral capsid. Dynamic light scattering and transmission electron microscopy of the DNA aptamer-decorated artificial viral capsid showed the formation of spherical assemblies with a diameter of approximately 50-150 nm. The artificial viral capsid was selectively internalized into the Burkitt lymphoma cell line, Daudi, and doxorubicin complexed with the capsid selectively killed Daudi cells.