Biology subjects

Taguchi, Y.

Publications and source records attributed to Taguchi, Y..

8 recordsLinked to original sources

bioRxiv2025

Flow-driven construction of capillary-scale vessels with predefined geometries in natural hydrogels

Forming capillary networks with predefined geometries is a critical challenge in engineering complex three-dimensional tissues in vitro. While bioprinting and microfluidic technologies have enabled vascular tissue fabrication, precise control over capillary-scale vascularization remains limited. In this study, we investigated vascular formation process along hydrogel microchannels to elucidate mechanisms governing capillary-scale lumen formation. Microchannels were fabricated by femtosecond laser ablation in collagen and fibrin hydrogels. We optimized multiphoton lithography parameters to fabricate microchannels within these hydrogels and analyzed vascular formation along the channels. Luminal vascular structures formed readily in 50-m channels, while vascular formation failed in 20-m channels under static conditions, suggesting a significant shift in endothelial organization at the cellular scale. Flow stimulation significantly promoted vessel formation through collective endothelial cell migration and adhesion, whereas static conditions induced endothelial-to-mesenchymal transition. These findings provide key insights into capillary-scale vascularization and contribute to the development of more complex architectures with predefined shapes.

bioengineering↗

bioRxiv2020

Phase transition of prion protein initiates proteinase K resistant amyloid formation

Prion diseases are characterized by accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert PrPC to proteinase-resistant PrP (PrP-res) in vitro; however, the intermediate steps involved in the spontaneous conversion remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation in the absence of PrPSc. We found that rPrP underwent liquid-liquid phase separation at the interface of the aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired proteinase-resistant amyloid accompanied by {beta}-sheet conversion, as confirmed by western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote liquid-liquid phase separation. Thus, structural conversion via liquid-liquid phase separation and liquid-solid phase transition are intermediate steps in the conversion of prions.

biochemistry↗

bioRxiv2019

Molecular dynamics simulation of local structural models of PrPSc reveals how codon 129 polymorphism affects propagation of PrPSc.

Prions are unconventional pathogen without nucleotide genome and their pathogenic properties are defined by the primary structure and the conformation of the constituent abnormal isoform (PrPSc) of prion protein (PrP). A polymorphic codon 129 of human PrP that is valine (V129) or methionine (M129) is particularly influential on properties of PrPSc, affecting transmission efficiencies and clinicopathological features. However, how the single residue is so influential has not been elucidated because the detailed structures of PrPSc have not been determined yet due to its incompatibility with high-resolution structural analysis. Previously we created an in-register parallel {beta}-sheet local structural model of human PrPSc encompassing residues 107 to 143 that seemed more compatible with V129 than M129, based on knowledge from -synuclein amyloids and an NMR-based model of the amyloid of Y145Stop mutant of PrP in the literature. Here, we created an M129-compatible local structural model of PrPSc. Severe destabilization of the model by G127V mutation was consistent with the protective effects of V127 polymorphism of human PrP against prions. It was highly sensitive to the length of the hydrophobic side chain of codon 129 and replacement of M129 with leucine or valine destabilized the structures. Interestingly, the U-shaped {beta}-arch which comprises M129 flexibly changed hydrophobic interaction networks inside the {beta}-arch depending on the interactions with the surrounding structures, whereas the previous model with V129 maintained the similar network patterns irrespective of the surroundings. The differences between the two models may explain influences of the codon 129 polymorphism on transmissions and properties of human prions.

molecular biology↗

bioRxiv2019

Conformation-dependent influences of hydrophobic amino acids in two in-register parallel β-sheet amyloids, an α-synuclein amyloid and a local structural model of PrPSc

Prions are unconventional pathogens that encode the pathogenic information in conformations of the constituent abnormal isoform of prion protein (PrPSc), independently of the nucleotide genome. Therefore, conformational diversity of PrPSc underlies the existence of many prion strains and species barriers of prions, although the conformational information is extremely limited. Interestingly, differences between polymorphic or species-specific residues responsible for the species/strain barriers are often caused by conservative replacements between hydrophobic amino acids. This implies that subtle differences among hydrophobic amino acids are significant for PrPSc structures. Here, we analyzed the influence of different hydrophobic residues on the structures of an in-register parallel {beta}-sheet amyloid of -synuclein (Syn) using molecular dynamics (MD) simulation, and applied the knowledge from the Syn amyloid to modeling a local structure of human PrPSc encompassing residues 107-143. We found that mutations equivalent to polymorphisms that cause transmission barriers substantially affect the stabilities; for example, the G127V mutation, which makes the host resistant to various human prion diseases, greatly destabilized the model amyloid. Our study demonstrates specifically how and in what structures hydrophobic residues can exert unique effects on in-register parallel {beta}-sheet amyloids and provides insights into the molecular mechanism of the strain diversity of prions and other pathogenic amyloids. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/758938v4_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1e55372org.highwire.dtl.DTLVardef@c98952org.highwire.dtl.DTLVardef@1566c06org.highwire.dtl.DTLVardef@ce1945_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

In silico evidence of unique behaviors of methionine in an in-register parallel beta-sheet amyloid, suggestive of possible contribution to strain diversity of amyloids.

Mechanism of strain diversity of prions is a long-standing conundrum, because prions consist solely of abnormal isoform of prion protein (PrPSc) devoid of genetic material. Pathogenic properties of prions are determined by conformations of the constituent PrPSc according to the protein-only hypothesis, and alterations to even a single residue can drastically change the properties when the residue is located at a critical position for the structure of PrPSc. Interestingly, methionine (Met) is often recognized as the polymorphic or species-specific residues responsible for species/strain barriers of prions, implying its unique influences on the structures of PrPSc. However, how it is unique is difficult to demonstrate due to lack of the detailed structures of PrPSc. Here we analyzed influences of Met substitutions on structures of an in-register parallel {beta}-sheet amyloid of -synuclein (Syn) by molecular dynamics (MD) simulation, to extrapolate the results to PrPSc. The MD simulation revealed that Met uniquely stabilized a U-shaped {beta}-arch of the Greek-key Syn amyloid, whereas other hydrophobic amino acids tended to destabilize it. The stabilizing effect of Met was attributable to the long side chain without C{beta} branching. Our findings exemplify specifically how and in what structure of an in-register parallel {beta}-sheet amyloid Met can uniquely behave, and are suggestive of its influences on structures of PrPSc and strain diversity of prions. We also discuss about relations between -helix propensity and local structures of in-register parallel amyloids.

molecular biology↗

bioRxiv2018

Mechanisms of strain diversity of disease-associated in-register parallel β-sheet amyloids and implications about prion strains.

The mechanism of strain diversity of prions still remains unsolved, because the investigation of inheritance and diversification of the protein-based pathogenic information demands identification of the detailed structures of abnormal isoform of prion protein (PrPSc), while it is difficult to purify for analysis without affecting the infectious nature. On the other hand, the similar prion-like properties are recognized also in other disease-associated in-register parallel {beta}-sheet amyloids including Tau and -synuclein (Syn) amyloids. Investigations into structures of those amyloids by solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances, because of their relatively small sizes and lack of post-translational modifications. We review the advances on those pathogenic amyloids, particularly Tau and Syn, and discuss their implications about strain diversity mechanisms of prion/PrPSc from the viewpoint that PrPSc is an in-register parallel {beta}-sheet amyloid. We also present our recent data of molecular dynamics simulations of Syn amyloid, which suggest significance of compatibility between {beta}-sheet propensities of the substrate and local structures of the template for stability of the amyloid structures. Detailed structures of the Syn and Tau amyloids are good surrogate models of pathogenic amyloids including PrPSc to elucidate not only the strain diversity but also their pathogenic mechanisms.

molecular biology↗

bioRxiv2018

G-quadruplex dynamics contribute to epigenetic regulation of mitochondrial function

Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Predicted G4-forming sequences in the mitochondrial genome are enriched on the heavy-strand and have been associated with formation of deletion breakpoints that cause mitochondrial disorders. However, the functional roles of G4 structures in regulating mitochondrial respiration in non-cancerous cells remain unclear. Here, we demonstrate that RHPS4, previously thought to be a nuclear G4-ligand, localizes primarily to mitochondria in live cells by mechanisms involving mitochondrial membrane potential. We find that RHPS4 exposure causes an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. At higher ligand doses, RHPS4 causes mitochondrial DNA (mtDNA) replication pausing and genome depletion. Using these different levels of RHPS4 exposure, we describe discrete nuclear gene expression responses associated with mitochondrial transcription inhibition or with mtDNA depletion. Importantly, a mtDNA variant with increased anti-parallel G4-forming characteristic shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-structure mediated. Thus, we demonstrate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells and describe the first molecule that differentially recognizes G4 structures in mtDNA.

molecular biology↗

bioRxiv2017

PrPSc-induced conformational changes and strain-specific structures of PrPSc revealed by Disulfide-crosslink scanning

There exist many phenotypically-varied prion strains, like viruses, despite the absence of conventional genetic material which codes the phenotypic information. As prion is composed solely of the pathological isoform (PrPSc) of prion protein (PrP), the strain-specific traits are hypothesized to be enciphered in the structural details of PrPSc. Identification of the structures of PrPSc is therefore vital for the understanding of prion biology, though they remain unidentified due to the incompatibility of PrPSc with conventional high-resolution structural analyses. Based on our previous hypothesis that the region between the first and the second -helix (H1[~]H2) and the distal region of the third helix (Ctrm) of the cellular isoform of PrP (PrPC) have important roles for efficient interactions with PrPSc, we created series of mutant PrPs with two cysteine substitutions (C;C-PrP) which were systematically designed to form an intramolecular disulfide crosslink between H1[~]H2 and Ctrm and assessed their conformational changes by prions: Specifically, a cysteine substitution in H1[~]H2 from 165 to 169 was combined with cysteine-scanning along Ctrm from 220 to 229. C;C-PrPs with the crosslinks were expressed normally with the similar glycosylation patterns and subcellular localization as the wild-type PrP albeit with varied expression levels. Interestingly, some of the C;C-PrPs converted to the protease-resistant isoforms in the N2a cells persistently infected with 22L prion strain, whereas the same mutants did not convert in the cells infected with another prion strain Fukuoka1, indicating that local structures of PrPSc in these regions vary among prion strains and contribute to prion-strain diversity. Moreover, patterns of the crosslinks of the convertible C;C-PrPs implied drastic changes in positional relations of H1[~]H2 and Ctrm in the PrPSc-induced conformational changes by 22L prion. Thus, disulfide-crosslink scanning is a useful approach for investigation of strain-specific structures of PrPSc, and would be applicable to other types of amyloids as well.

molecular biology↗