Biology subjects

Fukuda, Y.

Publications and source records attributed to Fukuda, Y..

17 recordsLinked to original sources

bioRxiv2026

A novel metalloprotease family discovered through a tardigrade

Tardigrades possess numerous proteins with poorly understood functions, including candidates proposed to have been acquired through horizontal gene transfer. Here, we characterized RvY_14254 from Ramazzottius varieornatus, previously designated ATPM5, whose homologs are widely distributed across bacteria and eukaryotes but remain largely uncharacterized. We determined the crystal structure of the protein, hereafter termed RvPeti, at 1.35 [A] resolution. RvPeti adopts a compact, C-shaped architecture with no close structural homologs in the Protein Data Bank and contains a conserved His-Asp-Asp (HDD) metal-binding motif within a deep surface pocket. Structural comparison revealed partial similarity of the active-site architecture to that of metalloproteases, including pseudoalterin. Anomalous diffraction analyses demonstrated that the HDD site can accommodate Zn2+ and Mn2+. Biochemical assays showed that RvPeti exhibits Zn2+ dependent proteolytic activity, which was strongly reduced by a metal chelating reagent and by alanine substitutions of the metal-coordinating residues. In contrast, only weak esterase activity was detected, with the substrates tested. These results identify RvPeti as a founding member of a previously uncharacterized family of metalloproteases and provide a structural framework for investigating the physiological and evolutionary roles of this widespread protein family.

biochemistry↗

bioRxiv2026

Purification-free cryo-EM reveals endogenous assemblies from minute tardigrade samples

Tardigrades are minute animals renowned for environmental stress tolerance, and their remarkable biology has motivated extensive molecular studies using biochemical and biophysical approaches. Yet the native structures of endogenous macromolecular assemblies in tardigrades remain largely inaccessible, in part because only limited biological material is available. Here we show that purification-free single-particle cryo-EM, guided by long-read cDNA sequence assignment, can recover multiple endogenous assemblies directly from crude extract of a wild-derived Macrobiotus cf. shonaicus culture. We reconstructed a dimeric vitellogenin (Vg) particle at 3.10 [A] resolution, a previously uncharacterized tardigrade ferritin at 3.09 [A] resolution, and a low-resolution ribosome map. The Vg particle is a glycosylated and metal-containing assembly with structural evidence of proteolytic processing and contains lipid-like densities occupying a broad chamber whose walls include N-linked glycans. The ferritin structure revealed a divergent C-terminal helix that lines a mineral-like density observed at a position corresponding to previously described mineralization sites in ferritin-family cages. These results establish purification-free cryo-EM as a route to structural analysis of endogenous macromolecular assemblies from minute animal samples. In tardigrades, this approach provides a foundation for future state-dependent structural studies, including desiccation, rehydration and reproduction.

biochemistry↗

bioRxiv2026

Cryo-EM structures of a neofunctionalized tardigrade peroxiredoxin specialized for nucleic acid binding

Some terrestrial tardigrades can endure severe oxidative stress in part through their gene-expanded repertoire of antioxidant proteins. However, among these antioxidant proteins, RvPrxL, a peroxiredoxin (Prx)-like protein from Ramazzottius varieornatus strain YOKOZUNA-1, is unusual in that the catalytic cysteine is replaced by glutamate, apparently incapacitating canonical peroxidase function. In this study, we investigated the structure and function of this atypical Prx. Biochemical assays demonstrated that RvPrxL completely lacks canonical peroxidase and chaperone activities. Cryo-EM analysis revealed a unique 20-mer structure of RvPrxL. We also observed that the tardigrade-specific N-terminal region of RvPrxL helps retain the high-order oligomeric assembly even in the presence of highly concentrated hydrogen peroxide. The N-terminal region also promotes nuclear localization of RvPrxL and mediates binding to nucleic acids including nuclear RNAs. Furthermore, combining a standard cryo-EM method and a new approach in which cell Lysates are Applied Directly on cryo-EM Grids (LApDoG), we visualized two distinct nucleic acid binding modes of RvPrxL, termed "on-ring" and "in-ring", which likely reflect distinct physiological roles or modes of action. Collectively, these findings show that RvPrxL has been neofunctionalized to interact with nucleic acids in the nucleus, highlighting unexpected functional diversification of antioxidant proteins in tardigrades.

biochemistry↗

bioRxiv2026

Genome compartments guide protamine replacement and genome stability during spermiogenesis

Compartment-scale genome organisation persists in mammalian sperm, yet how histone-protamine replacement is orchestrated in space and time during spermiogenesis remains unclear. Here we combine stage-resolved purification of mouse spermatids with spike-in-normalised ATAC-seq and PRM1 CUT&Tag to map chromatin accessibility and protamine incorporation across spermiogenesis. We uncover a transient, genome-wide hyper-accessible phase coincident with replacement that is uncoupled from transcription and suppressed in catalytic PHF7-mutant spermatids. PRM1 loading initiates within accessible A-compartment chromatin and later spreads across both A- and B-compartments, whereas protamine-null mice demonstrated that PRM1/ PRM2 deficiency selectively destabilises A-compartment closure. Sequencing of short DNA fragments from Prm1/Prm2 dosage-reduced epididymal sperm reveals early enrichment of breakage at protamine-targeted A-compartment regions, which dissipates as fragmentation becomes genome-wide during epididymal transit. Together, our data place histone-protamine replacement within a compartment-centred framework that links 3D genome domains to the timing, targeting and integrity of the sperm genome.

genomics↗

bioRxiv2025

A copper-dependent, redox-based hydrogen peroxide perception in plants

Redox-related molecules, such as quinones and reactive oxygen species (ROS), are important signaling molecules for all living organisms. A plant-specific leucine rich-repeat receptor-like kinase (LRR-RLK) CANNOT RESPOND TO DMBQ 1 (CARD1), also known as HYDROGEN-PEROXIDE-INDUCED Ca2+ INCREASES (HPCA1), perceives both quinones and ROS, but knowledge on key structural features is unknown. Here, we determine the structure of the CARD1 ectodomain and uncover its unique features. Structural studies, coupled with genetics and biochemical analysis, demonstrated that previously identified unique cysteine residues are not essential for signal perception in CARD1. Interestingly, CARD1 harbors a copper ion on the surface of the ectodomain via histidine-coordination that is crucial for hydrogen peroxide signaling. Our work reports the first copper-dependent redox receptor and highlights a unique perspective between a receptor and non-peptide stimuli during their perception.

plant biology↗

bioRxiv2025

Frontal midline theta power accounts for inter-individual differences in motor learning ability

Recent neurophysiological studies have demonstrated that frontal midline theta (FMT) activity plays a significant role in motor learning. One of the key challenges in motor learning is to understand the interindividual variability in learning proficiency rates, yet the underlying neural mechanisms remain unclear. To address this open question, this study recorded electroencephalogram activity during a visuomotor tracking task to investigate whether modulation of FMT power and the consistency of theta phase during motor preparation could explain individual differences in learning proficiency. We found a significant positive correlation between increased FMT power during motor preparation and learning proficiency rates. Specifically, individuals with greater FMT power exhibited faster learning rates. In contrast to this, no significant correlation was observed between the consistency of theta phase during motor preparation and learning proficiency. Together, these findings highlight that the FMT power, rather than phase synchrony, is closely associated with motor learning efficiency. This study provides a novel perspective for understanding the causes of individual differences in motor learning and further corroborates the previous evidence showing FMT power contributes to motor learning processes.

neuroscience↗

bioRxiv2024

Somatic mtDNA Mutation Burden Shapes Metabolic Plasticity in Leukemogenesis

The role of somatic mitochondrial DNA (mtDNA) mutations in leukemogenesis remains poorly characterized. To determine the impact of somatic mtDNA mutations on the process, we assessed the leukemogenic potential of hematopoietic progenitor cells (HPCs) from mtDNA mutator mice (Polg D257A) with or without NMyc overexpression. We observed a higher incidence of spontaneous leukemogenesis in recipients transplanted with heterozygous Polg HPCs and a lower incidence of NMyc-driven leukemia in those with homozygous Polg HPCs compared to controls. Although mtDNA mutations in heterozygous and homozygous HPCs caused similar baseline impairments in mitochondrial function, only heterozygous HPCs responded to and supported altered metabolic demands associated with NMyc overexpression. Homozygous HPCs showed altered glucose utilization with pyruvate dehydrogenase inhibition due to increased phosphorylation, exacerbated by NMyc overexpression. The impaired growth of NMyc-expressing homozygous HPCs was partially rescued by inhibiting pyruvate dehydrogenase kinase, highlighting a relationship between mtDNA mutation burden and metabolic plasticity in leukemogenesis. TEASERSomatic mtDNA mutations as drivers of metabolic change in the development of leukemia.

cancer biology↗

bioRxiv2023

Smart Parallel Automated Cryo Electron tomography

In situ cryo electron tomography enables investigation of macromolecules in their native cellular environment. Data collection, however, requires an experienced operator and valuable microscope time to carefully select targets for tilt series acquisition. Here, we developed a workflow using machine learning approaches to fully automate the entire process, including lamella detection, biological feature segmentation, target selection, and tilt series acquisition, all without the need for human intervention.

biophysics↗

bioRxiv2023

Isolation of stage-specific spermatogenic cells by dynamic histone incorporation and removal in spermatogenesis.

Due to the lack of an in vitro spermatogenesis system, studies on mammalian spermatogenesis require the isolation of specific germ cell populations for further analyses. BSA gradient and elutriation have been used for several decades to purify testicular germ cells; more recently, fluorescence-activated cell sorting (FACS) has become popular. Although each method has its advantages and disadvantages and is used depending on the purpose of the experiment, reliance on FACS is expected to be more prevalent because fewer cells can be managed. However, the currently used FACS method for testicular germ cells relies on karyotypic differences via DNA staining. Thus, it remains challenging to separate post-meiotic haploid cells (spermatids) according to their differentiation stage despite significant variations in morphology and chromatin state. In this study, we developed a method for finely separating testicular germ cells using VC mice carrying fluorescently tagged histones. This method enables the separation of spermatogonia, spermatocytes, and spermatids based on the intensity of histone fluorescence and cell size. Combined with a DNA staining dye, this method separates spermatids after elongation according to each spermiogenic stage. Although the necessity for a specific transgenic mouse line is less versatile, this method is expected to be helpful for the isolation of testicular germ cell populations because it is highly reproducible and independent of complex cell sorter settings and staining conditions.

developmental biology↗

bioRxiv2022

PPIX-binding Proteins Reveal Porphyrin Synthesis and Ferroptosis Link

All aerobic organisms require the cofactor heme to survive, but its synthesis requires formation of a potentially toxic intermediate protoporphyrin IX (PPIX). Little is known about the extent of PPIXs cellular interactions. Here, we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics with PPB identified common proteins among a diverse panel of mammalian cell lineages. Pathway and quantitative difference analysis revealed PPB-bound proteins related to iron and heme metabolism and suggested that these processes might be altered by heme/porphyrin synthesis. We show that increased heme/porphyrin synthesis in cells promotes ferroptosis that is pharmacologically distinct from canonical ferroptosis driven by erastin, an inhibitor of the cystine/glutamate antiporter. Proteomic data derived from PPB revealed an interactor, PRDX3, a mitochondrial peroxidase, that modulated heme/porphyrin biosynthesis driven ferroptosis. Consistent with a role in porphyrin-induced ferroptotic death targeted gene knockdown of PRDX3, but not peroxidases, PRDX1 or 2, enhanced porphyrin-induced ferroptotic death. The relationship between increased heme/porphyrin synthesis and ferroptosis was also found in a ferrochelatase-deficient T-lymphoblastoid leukemia cell line, suggesting potential strategy for treating certain cancers. We demonstrate that when the PPB probe is coupled with unbiased proteomics a previously unreported relationship between heme/porphyrin synthesis, and ferroptosis was discovered.

biochemistry↗

bioRxiv2021

Computational capability of ecological dynamics

Ecological dynamics is driven by an ecological network consisting of complex interactions. Information processing capability of artificial networks has been exploited as a computational resource, yet whether an ecological network possesses a computational capability and how we can exploit it remain unclear. Here, we show that ecological dynamics can be exploited as a computational resource. We call this approach "Ecological Reservoir Computing" (ERC) and developed two types of ERC. In silico ERC reconstructs ecological dynamics from empirical time series and uses simulated system responses as reservoir states, which predicts near future of chaotic dynamics and emulates nonlinear dynamics. The real-time ERC uses population dynamics of a unicellular organism, Tetrahymena thermophila. Medium temperature is an input signal and changes in population abundance are reservoir states. Intriguingly, the real-time ERC has necessary conditions for reservoir computing and is able to make near future predictions of model and empirical time series.

ecology↗

bioRxiv2021

Aqueous extract of Phyllanthus niruri protects against severe malaria by blocking erythrocyte invasion and modulating the host immune response

Plasmodium falciparum parasites are the major cause of malaria across Africa. Due to the appearance of multi-drug resistant parasites, new antimalarial drugs are needed. The medicinal plant Phyllanthus niruri is being used to treat fever and other symptoms of malaria in Nigeria; however, little is known about its antimalarial mechanisms. Here, we show that aqueous extract of P. niruri (PE) has multiple antimalarial effects, including anti-parasitic and host immunomodulatory activity. We found that co-culture of P. falciparum with PE drastically reduced parasite number, but PE did not inhibit parasite development or rupture; rather, it blocked erythrocytes invasion. In addition, we identified Astragalin as one of the antimalarial compounds which are contained in PE. Moreover, we found that PE suppresses the inflammatory activity and apoptosis of immune cells (T cells) and astrocytes and neurons in the central nervous system (CNS). Furthermore, we confirmed that oral administration of PE to mice suppressed parasite growth, excessive inflammation, CNS dysfunction, and the development of experimental cerebral malaria in an in vivo murine malaria model. Our findings demonstrate that PE has multiple effects on malaria progression, targeting both parasite and host cells.

pathology↗

bioRxiv2021

Serum hormone levels in infertile women between conceived with and without hormone replacement therapy: Safety of estradiol and progesterone replacement therapy in frozen-thawed embryo transfer cycles for breast cancer survivors.

ObjectiveRecent advances in cancer treatment and reproductive medicine have made the post-treatment quality of life an important concern for cancer survivors. We aimed to evaluate the safety of sex hormone (estradiol and progesterone) replacement therapy (HRT) in women who conceived by assisted reproductive technology (ART) with hormone receptor-positive breast cancer. MethodsWe measured serum E2 and P4 levels at 4-10 weeks of gestation in women who conceived naturally or after timed intercourse or intrauterine insemination for infertility without HRT for luteal support (non-HR group; n=135). We conducted a retrospective comparison of the values from the non-HR group with those of women who conceived by ART with HRT for infertility (HR group; n=75). ResultsSerum E2 levels were significantly higher in the non-HR group than in the HR group at 5, 6, and 8 weeks of gestation. Similarly, serum P4 levels were significantly higher in the non-HR group than in the HR group at 4, 5, and 6 weeks of gestation. ConclusionsThis study suggests that in cancer reproductive medicine for hormone-dependent breast cancer survivors, HRT administered during the first trimester of a pregnancy after primary disease treatment may not increase the sex hormone levels to levels above those seen in spontaneous pregnancy.

cancer biology↗

bioRxiv2020

Fast transport of RNA granules by direct interactions with KIF5A/KLC1 motors prevents axon degeneration

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long distance transport of non-membrane enclosed organelles such as RNA granules is not known. Here we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts directly with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that binding of SFPQ to KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie-Tooth (CMT) Disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations show that non-membrane enclosed organelles can move autonomously and that replacing axonally translated proteins provides a therapeutic approach to axonal degenerative disorders.

cell biology↗

bioRxiv2019

Phytolacca americana PaGT2 is an ambidextrous polyphenol glucosyltransferase

The health benefits of polyphenols have attracted their use as potential therapeutic agents, food additives, and cosmetics. However, low water solubility of polyphenols limits their cell absorbability, obscuring further exploration. Glycosylation is known to enhance the solubility of polyphenols preserving their pharmacological properties. Here, we show that a uridine diphosphate (UDP) glucosyltransferase from Phytolacca americana (PaGT2) regioselectively catalyzes the transfer of glucose from UDP-glucose to stilbenoids such as piceatannol and flavonoids such as kaempferol. To understand the structure-function relationship of PaGT2, we determined the crystal structure of PaGT2 as well as PaGT2 complexed with donor analogue UDP-2-fluoro glucose and stilbenoid acceptor analogues. While only one conserved histidine residue is recognized as a catalytic residue in known UGTs, the crystal structures of PaGT2 suggested the presence of two catalytically active residues (His18 and His81) at two sides of the catalytic pocket. Although the single catalytic residue mutant His18Ala or His81Ala did not completely impair the glucosylation activity of the enzyme, the double mutant His18Ala/His81Ala failed to form glucoside products. These results showed that both catalytic residues in PaGT2 actively and independently catalyze glucosylation, hence we called PaGT2 as an ambidextrous UGT. The information from PaGT2 will be advantageous for the engineering of efficient biocatalysts for production of therapeutic polyphenols.

biochemistry↗

bioRxiv2019

Identification of bacteria involved in the decomposition of lignocellulosic biomass treated with cow rumen fluid by metagenomic analysis

Previously, pretreatment of plant biomass using rumen fluid systems was developed to decompose cell wall. However, microbes which involved in plant cell wall decomposition in this system have not been identified, because the conditions of this system are different from the in situ rumen environment. We investigated the bacteria involved in the decomposition of cellulose and hemicellulose in a waste paper with the rumen pretreatment system using shotgun metagenomic analysis with next generation sequencing. After pretreatment of waste paper, about a half of the cellulose and hemicellulose content was decomposed. Genes encoding for cellulase and hemicellulase were mainly found to belonging to Ruminococcus, Clostridium, and Exiguobacterium. This study shows that Clostridium and Exiguobacterium, which have not been identified as predominant genus involved in cellulose and hemicellulose decomposition, might be categorized as the main fibrolytic bacteria in this system.

microbiology↗

bioRxiv2017

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in ciliate Tetrahymena

SUMMARY STATEMENTOur study demonstrates compositional and structural differences of the nuclear pore complex between the functionally differentiated macronucleus and micronucleus within a single cytoplasm of ciliated protozoa.\n\nABSTRACTThe nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of about 30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type, or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally-active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins, and subcellular localization analysis of GFP fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on compositional and structural specificity of NPCs.

cell biology↗