Biology subjects

Uchiyama, T.

Publications and source records attributed to Uchiyama, T..

6 recordsLinked to original sources

bioRxiv2026

The Lower γ Region Ensures Unidirectional Rotation and Torque Generation in the Latter Half of the 80° Substep of F1-ATPase

F1-ATPase achieves unidirectional rotation of its {gamma} shaft through coordinated conformational cycling of the 3{beta}3 ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF1 lacking the lower half of the rotor shaft by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six pauses per turn, indicating the presence of an additional intermediate during rotation. This previously unreported intermediate dwell was observed at 40{degrees} between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps confined to the 40{degrees} transition between the intermediate and catalytic dwells. Analysis of the dwell-time distributions indicated an approximately zero free energy bias between these two states, consistent with thermally driven interconversion. Cryo-EM resolved three corresponding intermediates--binding, intermediate, and catalytic dwells--showing a major {beta} conformational change from 0{degrees} to 40{degrees}, but minimal {beta} rearrangement between 40{degrees} and 80{degrees}, suggesting that the {beta} power stroke is not operative in this interval. Analysis of the {gamma} rotational scheme indicates that the driving force changes across the 0-80{degrees} step: the 0-40{degrees} advance proceeds via a power stroke to an intermediate dwell, whereas the subsequent 40{degrees} transition to the catalytic dwell lacks a power stroke and is dominated by thermal fluctuations. Taken together, these findings suggest that the lower {gamma} region contributes to unidirectional rotation and torque generation specifically in the latter half of the 0-80{degrees} step, while the initial half can proceed even without contributions from the lower {gamma} region.

biophysics↗

bioRxiv2026

Structural basis for loss of covalent flavinylation in the H158Y mutant of pyranose oxidase

Pyranose oxidase from Phanerochaete chrysosporium (PcPOx) is a flavoprotein that forms a covalent 8-N3-histidyl-FAD linkage at His158. Here, we determined the crystal structure of the PcPOx H158Y variant at 1.69 [A] resolution and investigated its FAD occupancy and catalytic activity. Tyr158 adopts a rotamer conformation incompatible with covalent flavinylation, placing its phenolic O atom 8.7 [A] away from the FAD C8 atom. This conformation is stabilized through a Tyr158-Lys79 hydrogen bond. The additional mutation K79A abolishes this hydrogen bond, potentially freeing the Tyr158 residue for other conformations, but does not restore covalent flavin attachment. The H158Y variant retains substantial FAD occupancy and tetrameric assembly but shows markedly reduced oxidase and dehydrogenase turnover rates (less than 13% of WT). These results provide a structural explanation for the failure of tyrosine substitution to support covalent flavinylation in PcPOx and offer insights into requirements for engineering alternative covalent flavin linkages.

biochemistry↗

bioRxiv2025

Integration of Hematopoietic and Thymus-like Niches in a Human iPSC-derived Bone Marrow Organoid

Human bone marrow generates blood cells but critically lacks the thymic environment required for T cell development. Developing an integrated in vitro platform that reconstitutes both functions simultaneously remains a major challenge in regenerative and immune medicine. We asked whether a synthetic marrow could be engineered to provide both capacities. Using human induced pluripotent stem cells, we created self-organizing bone marrow organoids (iBMOs) that faithfully reproduced native stromal and vascular structures and, remarkably, supported robust thymus-like T cell differentiation. iBMOs directed hematopoietic progenitors toward functional T and dendritic cells. When engrafted into immunodeficient mice, they autonomously sustained human erythropoiesis and de novo bone formation in vivo. Single-cell transcriptomics revealed a complex niche architecture, including a hybrid cluster co-expressing stem, endothelial, and stromal markers, key to understanding this dual functionality. This bioengineered organoid-stromal system unifies bone marrow and thymic functions in a single human-derived platform, offering a scalable source of immune cells for adoptive cell therapies and regenerative applications, as well as a versatile model for studying human hematopoiesis and immunity.

systems biology↗

bioRxiv2025

Low-Background Cancer Imaging With a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a Targeting Moiety

Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes are insufficiently bioorthogonal, resulting in high background signals in non-target tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes bearing various sugar moieties, and discovered that HMRef-{beta}-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a kcat/Km value for HMRef-{beta}-D-fucose of 3.3 x 105/M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of {beta}-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probes access to the enzymes active site. In a proof-of-concept study, cancer cells were visualized with a minimal background in the mesentery of a mouse model of peritoneally disseminated human-ovarian-cancer-derived SKOV-3 cells, which endogenously express HER2, by using HMRef-{beta}-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.

bioengineering↗

bioRxiv2024

Two Stabiliser loci in Antirrhinum suppress the transposition of Tam3 without compromising transposase production

Antirrhinum majus has been known to exhibit occasional instabilities that are manifested as variegations and morphological chimeras. Stabiliser (St) is a historical locus that stabilizes phenotypically unstable or mutable traits in Antirrhinum. Here, we characterized two St loci, the previously described Old Stabiliser (OSt) and New Stabiliser (NSt), in Antirrhinum that specifically suppress the transposition of the Class II DNA transposable element, Tam3. Both St loci involve derivatives of Tam3 with unique structures: OSt has a pseudo-Tam3 copy whose 5-terminal region has been rearranged compared to the cognate Tam3 element, and NSt consists of two intact copies of Tam3 in a head-to-head orientation. Neither locus interferes with the production of the intact Tam3 transposase (TPase) or the nuclear import of TPase. Both OSt and NSt produce specific sRNAs from their 5 terminal regions containing multiple TPase binding motifs. These specific sRNAs could repress Tam3 transposition by interacting with the TPase binding motifs within the Tam3 element or with the TPase itself.

genetics↗

bioRxiv2018

Genetic basis of transgressive segregation in rice heading phenotypes

Transgressive segregation produces hybrid progeny phenotypes that exceed parental phenotypes. Unlike heterosis, extreme phenotypes caused by transgressive segregation are heritably stable. We examined transgressive phenotypes of flowering time in rice. Our previous study examined days to flowering (heading; DTH) in six F2 populations for which the parents had distal DTH, and found very few transgressive phenotypes. Here, we demonstrate that transgressive segregation in F2 populations occurred between parents with proximal DTH. DTH phenotypes of the A58 x Kitaake F2 progenies frequently exceeded those of both parents. Both A58 and Kitaake are japonica rice cultivars adapted to Hokkaido, Japan, which is a high-latitude region, and have short DTH. Among the four known loci required for short DTH, three loci had common alleles in A58 and Kitaake, and only the one locus had different alleles. This result indicates that there is a similar genetic basis for DTH between the two varieties. We identified five new quantitative trait loci (QTLs) associated with transgressive DTH phenotypes by genome-wide single nucleotide polymorphism (SNP) analysis. Each of these QTLs showed different degrees of additive effects on DTH, and two QTLs had epistatic effect on each other. These results demonstrated that genome-wide SNP analysis facilitated detection of genetic loci associated with the extreme phenotypes and revealed that the transgressive phenotypes were produced by exchanging complementary alleles of a few minor QTLs in the similar parental genotypes.

genetics↗