Biology subjects

Somiya, M.

Publications and source records attributed to Somiya, M..

7 recordsLinked to original sources

bioRxiv2026

Computational design of membrane fusion proteins

The fusion of two distinct biological membranes is an evolutionarily conserved process essential to cellular organization and physiology. Membrane fusion is driven by the refolding of fusogenic proteins into low-energy postfusion states that overcome the energetic barrier to bilayer merger. Here we report a computational method for the design of synthetic fusogens inspired by the architecture of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Using machine learning-guided protein design to extensively remodel backbone geometry and sequence, we generated heterodimeric SNARE-like assemblies that efficiently catalyze cell-cell membrane fusion. These minimal two-component fusogens exhibit substantially higher fusion activity than native multisubunit SNARE complexes. Structural and functional analyses identify the key determinants required for fusogenic activity and reveal a modularity that enables control of fusion through chemically induced heterodimerization. In addition to cell-cell fusion, the synthetic fusogens drive fusion between endoplasmic reticulum and mitochondrial membranes from human cells, demonstrating their potential as tools for programmable manipulation of intracellular membranes. Together, these results establish a general framework for the rational design of synthetic fusogens and expand the toolkit for engineering membrane dynamics in living systems.

biochemistry↗

bioRxiv2026

Programmable protein editing by split intein-mediated recombination

Biological regulation has long relied on modifying genes, transcripts, or protein abundance, yet direct rewriting of protein sequence after translation remains largely inaccessible. Here, we introduce a programmable protein editing platform based on split intein-mediated recombination, enabling precise excision and replacement of defined segments within mature proteins in living cells. By covalently exchanging a target segment with a donor-encoded sequence supplied in trans, this approach functions as a protein-level analogue of recombination, operating independently of transcription or translation. Using ultrafast and orthogonal split inteins, we demonstrate efficient protein recombination in mammalian cells, enabling single-amino acid substitutions, domain replacement, functional switching, and reprogramming of subcellular localization. This work establishes post-translational protein recombination as a general strategy for sequence-level control of protein function, expanding the conceptual and practical scope of biological editing beyond nucleic acids.

biochemistry↗

bioRxiv2023

Computational design of non-porous, pH-responsive antibody nanoparticles

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

bioengineering↗

bioRxiv2022

Verification of extracellular vesicle-mediated functional mRNA delivery via RNA editing

The secretion and delivery of mRNA by extracellular vesicles (EVs) may contribute to intercellular communications. Several reporter assays have been developed to quantify EV-mediated functional delivery of mRNA into recipient cells. However, mRNA delivery efficiency can often be overestimated by experimental artifacts, resulting in "pseudo-delivery" of reporter proteins rather than mRNA. In this study, we revealed that substantial amounts of reporter proteins expressed in donor cells are secreted into the medium and interfere with the reporter assay. To eliminate this pseudo-delivery, we established a functional RNA delivery assay that employs an RNA editing tool, enabling the verification of bona fide delivery of mRNA into recipient cells. The donor cells expressed a reporter gene containing a stop codon in a non-functional open reading frame. After EV-mediated delivery of reporter mRNAs to the recipient cells, guide RNAs and RNA editing enzymes (dCas13b-hADAR2 fusion proteins) correct the RNA sequence and induce the expression of functional reporter proteins in the recipient cells. Using this system, we showed that EVs containing alphavirus-derived replicon successfully delivered functional RNA and expressed the reporter proteins. The RNA delivery assay using RNA editing enables the precise analysis of EV-mediated mRNA delivery.

cell biology↗

bioRxiv2021

Engineering of extracellular vesicles for small molecule-regulated cargo loading and cytoplasmic delivery of bioactive proteins

Cytoplasmic delivery of functional proteins into target cells remains challenging for many biological agents to exert their therapeutic effects. Extracellular vesicles (EVs) are expected to be a promising platform for protein delivery; however, efficient loading of proteins of interest (POIs) into EVs remains elusive. In this study, we utilized small compound-induced heterodimerization between FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain, to sort bioactive proteins into EVs using the FRB-FKBP system. When CD81, a typical EV marker protein, and POI were fused with FKBP and FRB, respectively, rapamycin induced the binding of these proteins through FKBP-FRB interaction and recruited the POIs into EVs. The released EVs, displaying virus-derived membrane fusion protein, delivered the POI cargo into recipient cells and their functionality in the recipient cells was confirmed. Furthermore, we demonstrated that CD81 could be replaced with other EV-enriched proteins, such as CD63 or HIV Gag. Thus, the FRB-FKBP system enables the delivery of functional proteins and paves the way for EV-based protein delivery platforms.

bioengineering↗

bioRxiv2021

Reporter gene assay for membrane fusion of extracellular vesicles

AbstractExtracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

cell biology↗

bioRxiv2020

Real-time luminescence assay for cytoplasmic cargo delivery of extracellular vesicles

Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for assessment of the cargo delivery efficiency inside recipient cells have not been feasible. In this study, we developed an EV cargo delivery (EVCD) assay using a split luciferase called the NanoBiT system. Recipient cells expressing LgBiT, a large subunit of luciferase, emit luminescence when the EV cargo proteins fused with a small luminescence tag (HiBiT tag) that can complement LgBiT are delivered to the cytoplasm of recipient cells. Using the EVCD assay, the cargo delivery efficiency of EVs could be quantitatively measured in real time. This assay was highly sensitive in detecting a single event of cargo delivery per cell. We found that modification of EVs with a virus-derived fusogenic protein significantly enhanced the cytoplasmic cargo delivery; however, in the absence of a fusogenic protein, the cargo delivery efficiency of EVs was below the threshold of the assay. The EVCD assay could assess the effect of entry inhibitors on EV cargo delivery. Furthermore, using a luminescence microscope, the cytoplasmic cargo delivery of EVs was directly visualized in living cells. This assay could reveal the biological mechanism of the cargo delivery processes of EVs.

cell biology↗