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Uchihashi, T.

Publications and source records attributed to Uchihashi, T..

At least 19 recordsLinked to original sources

Silicon exaptation discovers antimicrobial peptides

Advances in machine learning (ML) have enabled the de novo design of synthetic proteins and peptides tailored for specific structural or functional properties. As ML becomes a central tool in forward design, we suggest that its value extends beyond optimizing known objectives. It can also lead to what we term here in this work as silicon exaptation, revealing unexpected functional properties that were never part of the original design goals. This reflects a principle common in biology, that repurposes traits where features evolved for one role can later enable unforeseen functions. In this context, these accidental findings are not constrained by preconceptions but arise from latent function emergence and the algorithm's gradual drift, a new process that can co-evolves with human curiosity, intuition, and interpretive reasoning, which can be used to recognize unexpected patterns and transform them into new discoveries. In this study, we discovered a set of peptides through silicon exaptation, in which the ML pipeline was originally optimized for structural characteristics unrelated to any antimicrobial functionalities. The peptides were originally designed to adopt defined secondary structures in response to environmental stimuli, with no antimicrobial properties intended or included in the training objectives. We systematically evaluated the antimicrobial potential of these peptides using broth microdilution assays and membrane integrity tests, identifying several candidates with potent and selective antibacterial activity without detectable cytotoxicity toward mammalian cells.

molecular biology↗

Metastable Dimeric Intermediate Drives Native-like Spidroin Fibrillogenesis, while Dehydration Promotes Cross-β Transition

The exceptional mechanical properties of spider dragline silk originate from hierarchical spidroin self-assembly, yet mimicking this process to generate native-like structures remains challenging. Here, we report a metastable dimeric intermediate as an essential molecular factor for fibrillogenesis. Small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed a compact core stabilized by solvent-excluded "dry interfaces" surrounded by extended random coil regions. Within the core, a subset of the polyalanine blocks is held by side-chain packing rather than by main-chain {beta}-sheets; the majority remain exposed in the surrounding coil regions. Upon lowering the denaturant concentration, these exposed polyalanine segments pair with their counterparts at a fibril end, forming intermolecular {beta}-sheets that act as linkers and drive the stepwise end-to-end polymerization of the dimeric particles. In solution, the resulting fibrils exhibit an ordered {beta}-structure in which the polypeptide chains run parallel to the fibril axis, in contrast to amyloid-like cross-{beta}, in which the strands lie perpendicular to it; this matches key features of native silk nanofibril architecture. However, X-ray diffraction (XRD) analysis demonstrates that these native-like nanofibrils undergo an effectively irreversible transition toward amyloid-like cross-{beta} structures upon drying. Native-like fibrils are thus achievable by self-assembly, but structural control is lost during the final dehydration step. Accordingly, we propose a general principle for decoupling self-assembly from dehydration: native-like nanofibrils are formed first, then dried under conditions that restrict molecular-chain mobility (for example, mechanical constraint or immobilization on a surface), which we expect to suppress thermodynamic relaxation into cross-{beta} structures.

biophysics↗

Sensory neuron dysfunction and hyperexcitability in dorsal root ganglia at disease onset in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder traditionally characterized by motor neuron degeneration, but emerging evidence indicates sensory system involvement. Despite reports of sensory abnormalities in some patients, the molecular and functional alterations in dorsal root ganglion (DRG) neurons remain insufficiently characterized. We investigated DRG pathology at disease onset in 12-week-old SOD1G93A mice using an integrated transcriptomic, morphological, and electrophysiological approach. RNA sequencing of lumbar DRG identified 35 differentially expressed genes, predominantly upregulated, enriched in oxidative stress-related and phagosome pathways. Comparative analysis with motor neuron transcriptomes revealed distinct gene expression profiles, indicating sensory neuron-specific molecular responses. Immunohistochemistry demonstrated reduced soma diameter in both A- and C-fiber DRG neurons. Nav channel colocalization increased for Nav1.7 in A fibers and Nav1.8 in both fiber types, whereas Nav1.6 was unchanged. Whole-cell patch-clamp recordings showed depolarized resting membrane potential, increased spike amplitude, and enhanced repetitive firing in A-fiber neurons, consistent with hyperexcitability, while C fibers showed no significant functional changes. These findings demonstrate early molecular, structural, and functional alterations in primary sensory neurons in ALS, supporting pathology beyond motor neurons and identifying sensory neuron excitability as a potential therapeutic target.

neuroscience↗

High-speed atomic force-Raman microscopy

A comprehensive understanding of the mechanisms underlying biological systems requires correlative analysis of multiple complementary molecular properties through multidimensional measurements. High-speed atomic force microscopy (HS-AFM) is a powerful tool for elucidating biomolecular structural dynamics at the single-molecule level with high spatiotemporal resolution. However, structural information alone is often insufficient for fully understanding the biological function mechanisms. Here, we report high-speed atomic force-Raman microscopy (HS-AFRM), which enables multimodal measurements combining video-rate structural imaging with chemical-bond analysis. Raman spectroscopy is a powerful, non-invasive technique that probes molecular vibrations to provide chemical information. We achieved several key technical developments that facilitated the seamless integration of HS-AFM and micro-Raman spectroscopy, allowing reliable correlative measurements of structural and chemical information. We validated the versatility of the developed system using representative samples, including two-dimensional materials and a protein. Furthermore, we demonstrate probing of changes in the surrounding environment, which are inaccessible by HS-AFM alone. Multimodal measurements incorporating fluorescence spectroscopy were also demonstrated as an additional practical extension. This multimodal approach substantially enhances the analytical capability of HS-AFM, providing a powerful platform for revealing correlated structural and chemical properties across diverse research fields.

biophysics↗

Dynamic organizational strategies of multidomain glycosyltransferases revealed by high-speed AFM and solution biophysics

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open-closed interdomain motion and substrate-dependent conformational compaction, indicating dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands.

biophysics↗

A single conserved residue in FlhA couples export gate activation to substrate specificity switching

FlhA is a core component of the bacterial flagellar type III secretion system that functions as both an export engine and a switch regulator of substrate specificity. How these two processes are mechanistically coupled remains unclear. Here we identify a single conserved residue in FlhA, Arg-391, as a key molecular node that links export gate activation to substrate specificity switching. The R391A substitution blocks the transition from hook-type to filament-type secretion, resulting in polyhooks lacking filaments. An intermolecular salt-bridge formation of Arg-391 with Glu-351 of an adjacent FlhA subunit stabilizes the filament-type FlhA ring. Suppressor mutations in FlhA and the ruler protein FliK restore this interaction, indicating that FliK promotes the intermolecular salt-bridge formation during export switching. Moreover, Arg-391 is required for efficient ATPase-independent activation of the export gate. Together, these findings reveal an electrostatic mechanism by which FlhA coordinates export gate activation and substrate specificity switching during flagellar assembly.

microbiology↗

A dual-domain chitinase mechanism enables marine bacteria tosense and localize sparse crystalline chitin in the ocean

Chitin is one of the most abundant biopolymers in the ocean, and its remineralisation underpins major pathways of marine carbon cycling. A central challenge for chitinolytic bacteria is to locate and remain associated with sparsely distributed crystalline chitin particles. Here, we show that the GH18 chitinase VpChi1 from Vibrio parahaemolyticus possesses a dual-domain architecture that enhances particle encounter and retention. Full-length VpChi1 displayed much higher catalytic efficiency than a CBD-truncated mutant at low chitin concentrations, whereas substrate inhibition occurred at higher concentrations. Single-molecule fluorescence imaging demonstrated that the additional C-terminal chitin-binding domain (CBD) enables selective binding to the hydrophobic surface of crystalline chitin and substantially prolongs enzyme residence time. Kinetic simulations using experimentally determined dissociation rates showed that this extended residence arises from CBD-assisted rebinding of the catalytic domain. High-speed atomic force microscopy confirmed that the CBD does not alter stepping velocity or travel distance along individual crystals, indicating that substrate inhibition likely results from CBD engagement with neighbouring particles rather than impaired motility. Screening of seawater and marine sediments across multiple depths identified Vibrio and Pseudoalteromonas species as dominant chitin degraders, many encoding GH18 chitinases with dual CBDs, whereas terrestrial bacteria generally lacked this architecture. We propose that this two-domain configuration confers an ecological advantage by increasing enzyme retention on particulate chitin and enabling sustained production of chitobiose, a potent chemotactic signal for marine bacteria. These findings reveal a mechanistic basis for how bacteria sense and exploit particulate chitin and highlight an adaptive strategy for resource acquisition in the ocean.

molecular biology↗

Energy conversion mechanism revealed by ATP-free and ATP-1 dependent walking of myosin V motor

The mechanical processes of ATPase protein motors are coupled to their ATPase reactions. The free energy released at a given step(s) of the ATPase cycle is converted into mechanical work. Therefore, it has long been thought that ATP is the energy source for mechanical work. Here, we show that the double-headed myosin V (M5) motor can perform ATP-free walking. In the presence of ADP, M5 bound to F-actin by both heads makes a forward step when the trailing head is detached by an external force. This forward step harnesses the strain energy in the leading (L) head, provided from the strong binding energy between the L-head and actin. Consequently, strain generation reduces the binding affinity, which manifests as sporadic, brief L-head detachment. Strain generation and brief L-head detachment also occur during ATP-dependent walking to similar extents as during ATP-free walking. These results suggest that the strong binding energy is also converted into mechanical work during ATP-dependent walking.

biophysics↗

Nanoscale visualization of Drosophila E-cadherin ectodomain fragments and their interactions using DNA origami nanoblocks

The adhesive function of cell surface proteins can be visually assessed through direct observation; however, the underlying structures that mediate adhesion typically remain invisible at the nanoscale level. This hinders knowledge on the diversity of molecular architectures responsible for cell-cell adhesion. Drosophila E-cadherin (DE-cadherin), a classical cadherin with a unique domain structure, demonstrates adhesive function; however, it lacks a structural model that explains its adhesion mechanism. In this study, we present a novel application of DNA origami technology to create a cell-free, flat environment in which full DE-cadherin ectodomains are anchored using SNAP-tags and biotin-streptavidin interactions. DNA origami was assembled into a 120 nm long block, bearing 5 or 14 biotin:streptavidin sites that were evenly spaced on one lateral face. DE-cadherin ectodomain fragments were attached via biotinylated SNAP-tags. These decorated DNA origami nanoblocks were subjected to transmission electron and high-speed atomic force microscopy, which revealed a hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain, suggesting a role in mechanical flexibility. We also observed interactions between DE-cadherin ectodomains via their membrane-distal portions on single DNA origami nanoblocks. We reconstituted an adhesion-like process via pairing DNA origami nanoblocks using DE-cadherin ectodomain interactions. Homophilic associations of functional DE-cadherin ectodomains between the paired DNA origami nanoblocks were visualized at the nanoscale, displaying strand-like molecular configurations, likely representing the extracellular cadherin repeats without regular arrays of structural elements. This study introduces a DNA origami-based platform for reconstituting and visualizing cadherin ectodomain interactions, with potential applications for a broader range of adhesion molecules. HighlightsO_LIDNA origami technology was applied to perform a structure-function study of cadherin. C_LIO_LIDNA origami nanoblocks decorated with DE-cadherin ectodomains were observed by TEM/HS-AFM. C_LIO_LIA hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain was revealed. C_LIO_LIAn adhesion-like process was mimicked via pairing two nanoblocks using DE-cadherin ectodomain interactions. C_LIO_LIHomophilic associations of DE-cadherin ectodomains between the nanoblocks were visualized at the nanoscale level. C_LI

molecular biology↗

Submolecular-scale Hairpin DNA Folding Dynamics Studied by High-Speed AFM with Optical Tweezers

Optical tweezers have contributed to elucidate the folding mechanisms associated with biomolecules. By combining single-molecule or super-resolution techniques, imaging can also be performed while measuring or inducing force coupling with biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this problem, here, we developed a technique that combines optical tweezers with high-speed atomic force microscopy (AFM). To solve the problem of incompatible instrumental configurations, we developed a unique optical tweezers measurement system that is specialized for high-speed AFM. Upon applying an external force to a synthesized DNA secondary structure, we successfully visualized the dissociation of the duplex structure. Furthermore, we succeeded in observing spontaneous reannealing of the duplex structure upon releasing the force, which demonstrates that the folding reaction can be reversibly controlled. We also reveal that along with duplex unfolding, a metastable secondary structure is generated and its topology changes transiently over time. The results indicate that this technique provides structural insights that cannot be obtained by conventional fluorescence techniques.

biophysics↗

Structural basis for the interaction between the bacterial cell division proteins FtsZ and ZapA

Cell division in most bacteria is regulated by the tubulin homolog FtsZ as well as ZapA, a FtsZ-associated protein. However, how FtsZ and ZapA function coordinately has remained elusive. Here we report the cryo-electron microscopy structure of the ZapA-FtsZ complex. The complex forms an asymmetric ladder-like structure, in which the double antiparallel FtsZ protofilament on one side and a single protofilament on the other side are tethered by ZapA tetramers. In the complex, the extensive interactions of FtsZ with ZapA cause a structural change of the FtsZ protofilament, and the formation of the double FtsZ protofilament increases electrostatic repulsion. High-speed atomic force microscopy analysis revealed cooperative interactions of ZapA with FtsZ at a molecular level. Our findings not only provide a structural basis for the interaction between FtsZ and ZapA but also shed light on how ZapA binds to FtsZ protofilaments without disturbing FtsZ dynamics to promote cell division.

molecular biology↗

A look beyond topography: transient phenomena of Escherichia coli cell division captured with high-speed in-line force mapping

Life on the nanoscale has been made accessible in recent decades by the development of techniques that are fast and non-invasive. High-speed atomic force microscopy (HS-AFM) is one such technique that has proven to shed light on elusive mechanisms involving single proteins. Extending HS-AFM to effortlessly incorporate mechanical property mapping while maintaining fast imaging speed allows us to look deeper than topography and reveal more details of the nanoscale mechanisms that govern life. Here, we present high-speed in-line force mapping (HS-iFM), which enables the recording of mechanical properties and topography maps with high spatiotemporal resolution. Employing this method, a detailed study of the dynamic nanoscale mechanical properties of living Escherichia coli bacteria reveals localized stiffening during division, intricate details of the division process, formation and diffusion of pores in the membrane, and the impact of depressurization of a cell. All of these phenomena were recorded with a frame time as low as 15 s and a spatial resolution of 5.5 nm/pixel in topography and 22 nm/pixel in force maps, allowing the capture of transient phenomena on bacterial surfaces in striking detail.

biophysics↗

Key role of P230 in the hinge region on the IgG architecture and function

Immunoglobulin G (IgG) is a molecule that plays an important role in biological defense; IgG molecules have been applied as drugs due to their high specificity for antigens and their ability to activate immunity via effector molecules on immune cells. On the other hand, the flexibility of the hinge region makes it difficult to apply conventional structural biology approaches due to its dynamic conformational changes, and the mechanism of action of the molecule as a whole has not been elucidated. Here, we introduced a deletion amino acid mutation in the hinge region to elucidate the role of the hinge region and its effect on the structure and function of the IgG molecule. Deletion of Pro230 resulted in the formation of a half-molecular in which the interaction between heavy chains was lost. We elucidated the mechanism of half-IgG formation by structural analysis using nuclear magnetic resonance (NMR) measurements and by disulfide quantification using peptide mapping using LC-MS/MS. For this purpose, a new NMR stable isotope labeling method was introduced. Finally, cell assay revealed that the IgG half-molecules have specific Fc{gamma}RI-mediated activity. This report provides new insights into the higher-order structure formation of IgG molecules and is expected to contribute to the elucidation of the molecular basis of the Fc{gamma} receptor-mediated activation mechanism of the immune system.

biophysics↗

Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motors

The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring--the upper part of the MS-ring--using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared to that of Salmonella such as tilt angle differences of the core domain and the {beta}-collar region, the decrease of the inter-subunit interaction between core domains, and altered electrostatic inner-surface. The residues potentially interact with other flagellar components, such as FliE and FlgB, are well structurally conserved in Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species. IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to advancing of bacterial flagellar biology.

biophysics↗

FliH and FliI help FlhA bring strict order to flagellar protein export in Salmonella

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The FlhA ring is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly, but the mechanism remains a mystery. Here, we report that the highly conserved GYXLI motif of FlhA is important for ordered protein export by the fT3SS. The fT3SS with the flhA(Y369A/R370A/L371A/I372A) (AAAA) or flhA(Y369G/R370G/L371G/I372G) (GGGG) mutation did not switch the substrate specificity at an appropriate timing of hook assembly. The A372V/T and G372V substitutions recovered the export switching function of the AAAA and GGGG mutants, respectively, in the presence but not in the absence of FliH and FliI, components of the flagellar ATPase complex. Interestingly, a filament-type substrate, FlgL, was secreted via the fT3SS with the AAAA or GGGG mutation during hook assembly in the absence of FliH and FliI but not in their presence. These observations suggest that FlhA requires the flagellar ATPase complex not only to efficiently remodel its ring structure responsible for the substrate specificity switching of the fT3SS but also to correct substrate recognition errors that occur during flagellar assembly.

microbiology↗

Ether phospholipids modulate somatosensory responses by tuning multiple receptor functions in Drosophila

Transient receptor potential (TRP) and PIEZO channels are known receptors for physical stimuli, such as temperature and mechanical touch, respectively, in sensory nerves. As these receptors are localized in the plasma membrane, the modulation of sensory receptor activity by plasma membrane lipids has recently attracted attention. In this study, we focused on ether phospholipids (ePLs), which are abundant in neurons, and analyzed their role in somatosensation using Drosophila as a model. Reduced mechanosensory behavior was observed with ePL synthesizing gene knockout or knockdown in mechano-sensitive PIEZO-expressing neurons. The activation of PIEZO channels was significantly augmented in the presence of ePLs. Furthermore, we observed that ePLs modulate the thermosensory behavior and reduce thermal threshold of the thermosensitive TRPA1 channels. Finally, we revealed that ePLs affect membrane tension and lipid order of the plasma membrane in culture cells. Our study identified ePLs as a modulator of multiple somatosensation modalities in Drosophila, which underscore the significance of functional interaction between membrane lipid and sensory channel proteins.

molecular biology↗

Intratumoral administration of recombinant murine interleukin-12 prevents tumor progression and bone invasion

BackgroundOral squamous cell carcinoma (OSCC) progression is accompanied by bone invasion. Therefore, maintaining oral function is necessary to regulate tumor progression. Also, interleukin-12 (IL-12), a well-known anti-tumor cytokine, can suppress osteoclast differentiation in vitro. Accordingly, this study evaluated the therapeutic effects of locally administered IL-12 in an immunocompetent mouse model with mandibular bone invasion mimicking clinical features. MethodsWe investigated anti-bone resorption effects using SCCVII subcutaneous and bone invasion models both in immunocompetent and athymic mice. Furthermore, we measured bone resorption using micro-computed tomography. ResultsIntratumoral injection of recombinant murine IL-12 (r-mIL-12) significantly prolonged immunocompetent mouse survival and suppressed tumor growth and bone resorption. Real-time PCR analysis revealed that interferon-gamma (IFN-{gamma}) and Fas ligand (FasL) were upregulated after r-mIL-12 administration, compared to control levels. However, when the athymic mouse bone invasion model was evaluated, r-mIL-12-mediated suppression of tumor growth and bone resorption were equivalent to those observed in the control group, highlighting the key role of T cells in the bone invasion. Conclusionsr-mIL-12 may represent a potent therapeutic agent for OSCC accompanied by bone invasion. SUMMARYIntratumoral injection of recombinant murine IL-12 showed anti-tumor and anti-bone resorption effects in an immunocompetent mouse bone invasion model through a T cell-dependent mechanism.

cancer biology↗

Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane

The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor-stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the two-transmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF-fliG fusion gene, which encodes an approximately 100 kDa protein, and the successfully production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region. We speculate that this flexibility plays a crucial role in facilitating the interaction between the cytoplasmic region of PomA and FliG to generate force. IMPORTANCEMS-ring is the initial structure to be assembled in flagellar motors. It comprises a complex two-ring (M and S) structure composed of 34 FliF molecules. We prepared a FliF-FliG fusion protein, which is directly involved in force generation. We observed it enabled the efficient formation of the MS-ring. The FliG portion that usually comprises the C-ring along with FliM and FliN displayed high flexibility likely due to the lack of FliM and FliN in the fusion protein. This study represents a significant milestone in the in vitro reconstruction of Na+-driven motor complexes.

biochemistry↗