Biology subjects

Moriwaki, Y.

Publications and source records attributed to Moriwaki, Y..

12 recordsLinked to original sources

bioRxiv2026

RGI-Toolkit: Differentiable Restraints for Controllable Biomolecular Structure Prediction

Diffusion-based models have substantially advanced biomolecular structure prediction. Within this framework, restraint-guided inference (RGI) corrects the denoiser output at each reverse-diffusion step using a differentiable restraint loss, allowing available experimental information, stereochemical requirements, or a specified target conformational state to be incorporated into predictions without retraining the model. Here, we present RGI-Toolkit, a general-purpose Python library that makes RGI available for a range of structure predictors. The toolkit consolidates restraint definition, atom selection, loss evaluation, and coordinate optimization into a shared, backend-agnostic engine, limiting predictor-specific code to a thin preprocessing adapter and a single hook in the denoising loop. Restraints on distances, angles, and dihedrals, on deviations from reference structures, and on user-defined reaction coordinates, together with ligand conformer restraints, are all specified in a common configuration format. The toolkit currently supports six predictors, including AlphaFold3. Across all six predictors, conformer restraints eliminated chirality and cis/trans errors in the predicted ligands and improved their local geometry, while geometric and custom restraints systematically shifted the sampled protein conformations toward the specified target states. RGI-Toolkit thus provides a unified framework for integrating experimental information and prior geometric knowledge into structure prediction and for generating biomolecular structures with specified conformational and stereochemical properties.

bioinformatics↗

bioRxiv2026

Distance-Restraint-Guided Diffusion Models for Sampling Protein Conformational Changes and Ligand Dissociation Pathways

Protein conformational dynamics and ligand binding processes are fundamental to biological function, yet their systematic sampling and thermodynamic characterization remain challenging. Here, we present a distance-restraint-guided inference method that extends AlphaFold3-like diffusion model frameworks to predict protein structures at specified conformational states. By restraining intergroup distances defined as center-of-mass distances between atom groups during the reverse diffusion process, our method enables systematic sampling along reaction coordinates without model retraining. We implemented this approach in Boltz-2 and demonstrated its effectiveness on three model proteins that undergo open-closed conformational transitions, as well as on a protein-peptide dissociation pathway. Compared with conventional approaches that induce conformational diversity by manipulating input multiple sequence alignments, our method achieved more uniform coverage of conformational space while maintaining high structural quality as assessed by both learning-based confidence metrics and stereochemistry-based validation. By combining distance-restrained sampling with molecular dynamics simulations, we constructed free energy landscapes and quantitatively estimated binding free energies. Altogether, our approach bridges deep learning-based structure prediction and physics-based simulations, providing an efficient strategy for characterizing the dynamics of biomolecules. The materials supporting this article, including the source code and Colab notebook are available at github (https://github.com/cddlab/boltz_restr).

biophysics↗

bioRxiv2025

ABCG transporters are involved in the accumulation of specialized metabolites in the oil bodies of Marchantia polymorpha

Bioactive specialized metabolites (SMs) are synthesized and sequestered in specific cellular compartments or organelles as a self-defense strategy against their intrinsic toxicity. Liverwort-specific oil bodies accumulate large amounts of SMs and contribute to chemical defense; however, the molecular mechanisms underlying SM sequestration in oil bodies remain largely unknown. Therefore, in this study, we focused on MpABCG1 and MpABCG36, which are ATP-binding cassette (ABC) protein family members localized to the oil bodies of liverwort Marchantia polymorpha. Sesquiterpene (thujopsene, chamigrene, and himachalane) accumulation was reduced in the Mpabcg1 and Mpabcg36 loss-of-function mutants. Notably, levels of the bisbibenzyls, marchantins C and A, were predominantly reduced in Mpabcg1, but not in Mpabcg36. Although the Mpabcg1 mutant formed a number of oil bodies labeled with mCitrine- MpSYP12B (oil body membrane marker) comparable to that of the wild-type, the number of oil bodies stained with BODIPY 493/503, which has an affinity for lipophilic SMs, was reduced. This finding suggests that MpABCG1 and MpABCG36 mutations affect SM accumulation in the oil body but have little impact on oil body formation. Overall, our results highlight the involvement of MpABCG1 and MpABCG36 in the accumulation of SMs and/or their precursors in liverwort oil bodies.

plant biology↗

bioRxiv2025

Predicting protein complexes in biosynthetic gene clusters

Biosynthetic gene clusters (BGCs) are contiguous genomic regions that encode diverse proteins responsible for natural product biosynthesis. These proteins collectively produce various secondary metabolites with complex chemical structure, including antibiotics and mycotoxins, yet the complete biosynthetic pathways have been experimentally resolved for only a limited number of compounds. Protein-protein interactions within BGCs have recently been recognized as key determinants of intermediate transfer, enzymatic regulation, and structural stability. However, many BGCs still contain proteins of unknown function that cannot be predicted by conventional sequence-based bioinformatics tools, hindering a comprehensive understanding of their biosynthetic pathways. To address this challenge, we built a high-throughput complex prediction pipeline by replacing AlphaFold3s multiple sequence alignment generation with a faster MMSeqs2. We systematically screened 487,828 protein pairs derived from 2,437 BGCs registered in the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database and predicted 15,438 heteromeric interactions with an ipTM [≥] 0.6. Among them, 1,390 protein pairs exhibited structural homology with an RMSD [≤] 2.0 [A]. These predictions highlight interesting molecular mechanisms involving proteins previously annotated as "uncharacterized" or "potentially dysfunctional". Our analysis further showed that the ipSAE metric can distinguish correct heterocomplex pairs when multiple functionally homologous proteins are present within a BGC. Overall, our computational analysis revealed molecular interaction networks among proteins encoded by each BGC and identified enzyme complexes that are likely functional only when assembled. These predicted complexes may represent previously unrecognized links in their biosynthetic pathways. The complete results are available in a reusable format at https://doi.org/10.5281/zenodo.17451667 to support future experimental validation.

bioinformatics↗

bioRxiv2025

Multi-objective optimization for designing structurally similar proteins with diverse sequences

Recent advances in artificial intelligence technologies have accelerated the development of computational protein design techniques. Although the structure of the designed proteins has been the primary focus, diversity of designed amino acid sequences is another important aspect of protein design. To address the trade-off between reducing sequence similarity and improving structural similarity, simultaneous optimization of these two objectives can be effective. We present a method that integrates ProteinMPNN with the multi-objective optimization algorithm NSGA-II to design proteins that retain high structural similarity to a target while exhibiting low sequence similarity. Using Top7 as a reference protein, we demonstrate that our approach improves sequence diversity compared to the original ProteinMPNN while maintaining comparable structural similarity.

bioinformatics↗

bioRxiv2025

Immune Niche Formation in Engineered Mouse Models Reveals Mechanisms of Tumor Dormancy

Residual tumor cells can persist in a dormant state during clinical remissions that may last decades. The mechanisms that lead to such growth control vs. eventual reactivation and macroscopic tumor outgrowth remain unclear. Here, we report data from a mouse model that reveals a key role of host immunity and the cellular and molecular mechanisms that control tumor dormancy. Abrogation of myeloid-specific TGF-{beta}RII expression (T{beta}RIImyeKO) resulted in an IFN-{gamma} rich immune microenvironment. IFN-{gamma} in turn elevated KLF4-mediated SLURP1 production in malignant cells, which is critical to the tumor cell quiescent state through interruption of fibronectin-integrin signaling pathways. The dormant tumor lesions were located in spatially localized immune niches rich in NK cells, cDCs, monocytes, and neutrophils, concomitant with tumor cell inactivation of NK cell immune surveillance through a CD200-CD200R1 mechanism. Our studies identify the IFN-{gamma}-KLF4-SLURP1 and CD200-CD200R1 axes as critical molecular drivers in tumor dormancy regulated by immune-tumor crosstalk. These insights provide enhanced mechanistic understanding of tumor dormancy in a mouse model suitable for further investigation of cancer treatment resistance and prevention of metastatic spread.

cancer biology↗

bioRxiv2025

Improving Stereochemical Limitations in Protein-Ligand Complex Structure Prediction

AlphaFold3 has revolutionized biology by enabling the prediction of protein complexes with various biomolecules, including small molecular ligands. However, the ligand structures predicted by the model often contain stereochemical errors. In this study, our comprehensive evaluation of AlphaFold3 and its clone model, Boltz-1, demonstrated significant limitations in ligand stereochemistry of their predicted structures, including chirality, bond, and angle geometries. To address the issue, we developed the restraint-guided inference method that applies stereochemical restraints during the reverse diffusion process. This approach perfectly reproduces the chirality specified in the input chemical structure and improves bond and angle geometries of ligands, while maintaining comparable performance in binding pose prediction. Our method provides a practical solution to the stereochemical errors in predicting protein-ligand complexes, thereby enhancing applications of structure prediction in structural biology and drug discovery.

bioinformatics↗

bioRxiv2024

Experimental method for creating skin with acquired appendage dysfunction

Mammalian skin appendages, such as hair follicles and sweat glands, are essential for both esthetic and functional purposes. Conditions such as burns and ulcers can lead to dysfunction or loss of skin appendages and result in hair loss and dry skin, posing challenges in their regeneration. Existing animal models are insufficient for studying acquired dysfunction of skin appendages without underlying genetic causes. This study aimed to develop more clinically relevant mouse models by evaluating two approaches: keratinocyte transplantation and grafting of skin at varying thicknesses. GFP-expressing keratinocytes were transplanted into ulcers on nude mice, leading to re-epithelialization with minimal skin appendages at 4 weeks after transplantation. However, the re-epithelialized area was largely derived from recipient cells, with the grafted cells contributing to only 1.31% of the area. In the skin grafting model, donor skin from GFP transgenic mice was grafted onto nude mice at three thicknesses: full thickness, 10/1000 inch, and 5/1000 inch. The grafted area of the 5/1000-inch grafts remained stable at 89.5% of its original size 5 weeks after transplantation, ensuring a sufficiently large skin area. The 5/1000-inch grafts resulted in a significant reduction in skin appendages, with a mean of only 3.73 hair follicles per 5 mm, compared with 69.7 in the control group. The 5/1000-inch skin grafting in orthotopic autologous transplantation also showed the achievement of skin surfaces with a minimal number of skin appendages. Therefore, a mouse model with skin grafting demonstrated stability in producing large areas of skin with minimal appendages. In conclusion, these two models with acquired skin appendage dysfunction and no underlying genetic causes provide valuable tools for researching skin appendage regeneration, offering insights into potential therapeutic strategies for conditions involving skin appendage loss.

bioengineering↗

bioRxiv2024

Optimization of an adeno-associated viral vector for keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

bioengineering↗

bioRxiv2023

Insights into stereoselective ring formation in canonical strigolactone: Discovery of a dirigent domain-containing enzyme catalyzing orobanchol synthesis

Strigolactones (SLs) are plant apocarotenoids with diverse functions and structures. The widespread canonical SLs, with distinctive structural variations in their tricyclic lactone known as the ABC-ring, are classified into two types based on the C-ring configurations. The steric C-ring configuration arises during the BC-ring closure downstream of carlactonoic acid (CLA), a biosynthetic intermediate. Most plants stereoselectively produce either type of canonical SLs, e.g., tomato (Solanum lycopersicum) produces orobanchol with -oriented C-ring. The mechanisms governing SL structural diversification are partly understood, with limited insight into the functional implications. Moreover, the precise molecular mechanism for the stereoselective BC-ring closure reaction remains unknown. Herein, we identified an enzyme called the stereoselective BC-ring-forming factor (SRF) from the dirigent protein (DIR) family, especially the DIR-f subfamily, whose biochemical function was previously unidentified, making it a pivotal enzyme in stereoselective canonical SL biosynthesis with the -oriented C-ring. We begin by confirming the exact catalytic function of the tomato cytochrome P450 SlCYP722C, which we previously demonstrated to be involved in the orobanchol biosynthesis [Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. Subsequently, we demonstrate that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid to form orobanchol. Our approach integrates experimental and computational methods, including SRF structure prediction and molecular dynamics simulations, to propose a catalytic mechanism based on the conrotatory 4{pi}-electrocyclic reaction for stereoselective BC-ring formation in orobanchol. The present study provides insight into the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

plant biology↗

bioRxiv2023

Comprehensive computational analysis of the SRK-SP11 molecular interaction underlying self-incompatibility in Brassicaceae using improved structure prediction for cysteine-rich proteins

Plants employ self-incompatibility (SI) to promote cross-fertilization. In Brassicaceae, this process is regulated by the formation of a complex between the pistil determinant S receptor kinase (SRK) and the pollen determinant S-locus protein 11 (SP11, also known as S-locus cysteine-rich protein, SCR). In our previous study, we used the crystal structures of two eSRK-SP11 complexes in Brassica rapa S8 and S9 haplotypes and nine computationally predicted complex models to demonstrate that only the SRK ectodomain (eSRK) and SP11 pairs derived from the same S haplotype exhibit high binding free energy. However, predicting the eSRK-SP11 complex structures for the other 100+ S haplotypes and genera remains difficult because of SP11 polymorphism in sequence and structure. Although protein structure prediction using AlphaFold2 exhibits considerably high accuracy for most protein monomers and complexes, 46% of the predicted SP11 structures that we tested showed < 75 mean per-residue confidence score (pLDDT). Here, we demonstrate that the use of curated multiple sequence alignment (MSA) for cysteine-rich proteins significantly improved model accuracy for SP11 and eSRK-SP11 complexes. Additionally, we calculated the binding free energies of the predicted eSRK-SP11 complexes using molecular dynamics (MD) simulations and observed that some Arabidopsis haplotypes formed a binding mode that was critically different from that of B. rapa S8 and S9. Thus, our computational results provide insights into the haplotype-specific eSRK-SP11 binding modes in Brassicaceae at the residue level. The predicted models are freely available at Zenodo, https://doi.org/10.5281/zenodo.8047768.

bioinformatics↗

bioRxiv2023

In vivo reprogramming of wound-resident cells generates skin with hair

Summary ParagraphMammalian skin appendages, such as hair follicles and sweat glands, are complex mini-organs formed during skin development1, 2. As wounds heal, the resulting scar tissue lacks skin appendages. The clinical regeneration of skin appendages is an ongoing challenge3, 4. Skin epithelial tissues have been regenerated in vivo by cellular reprogramming5, 6, but the de novo generation of skin appendages has not previously been achieved. Here, we show that transplantation of a type of epithelial cell and two types of mesenchymal cells, reprogrammed from adult mouse subcutaneous mesenchymal cells to mimic developing skin cells, resulted in the generation of skin-appendage-like structures. Furthermore, with the development of a new AAV serotype, in vivo reprogramming of wound-resident cells with the same reprogramming factors generates skin with de novo appendages in adult mice. These findings may provide new therapeutic avenues for skin regeneration and frequent aging-associated skin appendage disorders, such as hair loss and dry skin, and may extend to other tissues and organs. This study also provides the potential for de novo generation of complex organs in vivo.

cell biology↗