Biology subjects

Kato, T.

Publications and source records attributed to Kato, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Airway Epithelial SARS-CoV-2 Infectious and Repair Responses: Relationships to Age, Sex, and Post-COVID Pulmonary Syndromes

The long-term pulmonary sequelae of SARS-CoV-2 respiratory infections reflect infection severity, innate and adaptive immunity, and respiratory epithelial repair. This study investigated the acute and reparative responses as a function of age and sex in primary human bronchial epithelial (HBE) cultures utilizing a 14-day SARS-CoV-2 infection protocol. SARS-CoV-2 infection peaked at 3 days post-infection (dpi) with an [~] 2 log titer suppression at 14 dpi. SARS-CoV-2 infection induced interferon, interferon-induced gene, and cell damage responses. No age- or sex-dependent effects on SARS-CoV-2 infection were detected. Airway epithelia repaired to an abnormal mucus metaplastic/inflammatory state that reflected potentially beneficial and adverse consequences at 14 dpi. Repair processes were infection severity-dependent, not sex-dependent, and were more robust in young donor cultures. Analyses of long-COVID subjects with persistent pulmonary fibrosis or persistent bronchitic airway diseases exhibited expression of HBE 14 dpi failed repair gene signatures, including ISG gene signatures. Human airway epithelial repair post-SARS-CoV-2 is prolonged and incomplete in vitro over 14 days, and persistently abnormal repair may contribute to phenotypes of people with long-COVID pulmonary syndrome.

cell biology↗

bioRxiv2025

The Na+-pumping mechanism driven by redox reactions in the NADH-quinone oxidoreductase from Vibrio cholerae relies on dynamic conformational changes

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is a key respiratory enzyme in many marine and pathogenic bacteria that couples electron transfer to Na+-pumping across the membrane. Earlier X-ray and cryo-EM structures of Na+-NQR from Vibrio cholerae suggested that the subunits harboring redox cofactors undergo conformational changes during catalytic turnover. However, these proposed rearrangements have not yet been confirmed. Here, we have identified at least five distinct conformational states of Na+-NQR using: mutants that lack specific cofactors, specific inhibitors or low-sodium conditions. Molecular dynamics simulations based on these structural insights indicate that 2Fe-2S reduction in NqrD/E plays a crucial role in triggering Na+ translocation by driving structural rearrangements in the NqrD/E subunits, which subsequently influence NqrC and NqrF positioning. This study provides the first structural insights into the mechanism of Na+ translocation coupled to electron transfer in Na-NQR.

biochemistry↗

bioRxiv2025

Hypomorphic Lig4 gene mutation in mice predisposes to Th1-skewing intestinal inflammation

Dysregulation of DNA double-strand break (DSB) repair leads to adaptive immunodeficiency, whereas the remaining lymphocytes are aberrantly activated and provoke inflammations. However, no model mice were available to consistently manifest inflammation under defective DSB repair. We generated mutant mice carrying a missense mutation p.W447C in the gene encoding DNA ligase IV (LIG4), critical for DSB repair. Lig4W447C/W447C mice showed growth retardation and severe intestinal inflammations under adaptive immunodeficiency. The inflammations were featured by marked infiltration of T helper type 1 (Th1) cells and macrophages and was dependent on lymphocytes. When Ifng was deleted, Th2 and Th17 instead of Th1 cells drove the inflammations. Lig4W447C/W447C mice showed expansion of oligoclonal T cells with T cell receptor repertoire skewed towards more proximal 3 V and 5 J gene segments. Thus, our novel hypomorphic Lig4 mutant mice show that defective DSB repair leads to Th1-dependent intestinal inflammations under severe adaptive immunodeficiency.

immunology↗

bioRxiv2025

RXR agonist S169 inhibits HBV/HDV entry in vitro by disrupting KIF4-dependent NTCP trafficking.

Chronic hepatitis B (HBV) and hepatitis D (HDV) viral infections pose serious global health challenges, and the lack of curative therapies calls for the development of new antiviral approaches. Recently, we have identified the Kinesin Family Member 4 (KIF4) as a crucial regulator of the surface transport for NTCP (HBV/HDV entry receptor). Our research has shown that the pan-RXR agonist, Bexarotene, suppresses KIF4 expression and inhibits HBV/HDV infections. However, the clinical use of Bexarotene is limited by poor physicochemical and pharmacokinetic (PK) properties and significant side effects. Here we investigated chemically diverse RXR modulators with tuned activity, and identified the RXR agonist S169 as a novel HBV/HDV entry inhibitor. S169 effectively blocks HBV and HDV infections in primary human hepatocyte (PXB) cultures without evident cytotoxicity. S169s suppressive effect on HBV infection was confirmed by reduction of secreted HBsAg and HBeAg, as well as inhibition of HBV cccDNA, pregenomic RNA, and intracellular HBV DNA. Mechanistically, S169 suppressed FOXM1-mediated KIF4 expression, leading to decreased NTCP surface levels and a marked decrease in HBV preS1 peptide binding to the hepatocyte cell surface. S169 is reported to exhibit enhanced oral bioavailability, favorable PK parameters, lower toxicity compared to Bexarotene. Interestingly, S169 induced a modest inhibition of NTCP-mediated bile acid transport up to 10 M, which remained unchanged at higher concentrations. Furthermore, S169 significantly impeded HBV spread in HBV infected long-time cultured PHHs (PXB). We speculate that S169 can be a promising seed for the development of novel oral HBV and HDV antiviral therapeutics.

microbiology↗

bioRxiv2025

Discovery of The Clinical Candidate S-892216: A Second-Generation of SARS-CoV-2 3CL Protease Inhibitor for Treating COVID-19

The coronavirus disease 2019 (COVID-19) pandemic crisis has been mitigated by worldwide efforts to develop vaccines and therapeutic drugs. However, there remains concern regarding public health and an unmet need for therapeutic options. Herein, we report the discovery of S-892216, a second-generation SARS-CoV-2 3C-like protease (3CLpro) inhibitor, to treat COVID-19. S-892216 is a reversible covalent 3CLpro inhibitor with highly potent antiviral activity and an EC50 value of 2.48 nM against SARS-CoV-2 infected cells. Structure-based design of a covalent modifier for compound 1 revealed that introducing a nitrile warhead increased 3CLpro inhibition activity by 180-fold. Subsequent optimization efforts yielded S-892216, which combined a favorable pharmacokinetic profile and high off-target selectivity. S-892216 exhibited antiviral activity against diverse SARS-CoV-2 variants, with no cross-resistance to major mutations reducing antiviral activities of nirmatrelvir and ensitrelvir. In SARS-CoV-2-infected mice, S-892216 inhibited viral replication in the lungs similar to ensitrelvir, although at a 30-fold lower dose.

biochemistry↗

bioRxiv2025

Efficient identification of antimalarials with novel mechanism of action via multi-sample transcriptomic profiling

Malaria poses a significant health burden to endemic countries, necessitating the development of new antimalarials with novel mechanisms of action to combat emerging drug resistance. However, target identification methods for antimalarial compounds selected through phenotypic screening are currently limited, which has been a major obstacle in the field of antimalarial drug discovery. Here, we aim to address this need using perturbation transcriptomics to efficiently select antimalarial compounds targeting new pathways or molecules. The method involves comparing the transcriptomic changes by drug perturbation in the asexual blood stage of the malaria parasite Plasmodium falciparum. Using this method, we identified several compounds that exhibited distinct transcriptomic profiling from a diverse set of antimalarials with known modes of action, indicating their potential to target different pathways. In addition, we successfully identified a novel histone deacetylase inhibitor using this approach and chemogenetically validated its target through in vitro evolution. These results highlight the effectiveness of this platform in accelerating antimalarial drug discovery.

microbiology↗

bioRxiv2025

Immunological characterization of peritoneal exudate cells in liver cirrhosis patients

BACKGROUND AND AIMSLiver cirrhosis (LC) is the end stage of liver fibrosis caused by various chronic liver diseases. Patients with LC often develop ascites containing peritoneal exudate cells (PECs). However, those cells have not been fully immunologically characterized. In this study, we clarify immune cell profiles of PECs from patients with LC. APPROACH AND RESULTSPECs were collected from patients with LC or patients receiving continuous ambulatory peritoneal dialysis (CAPD) as a non-cirrhotic control and subjected to single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq) and flowcytometry analyses. Analysis of scRNA-seq revealed that dendritic cells (DCs) and macrophages were major populations in CAPD patient-derived PECs, while those cells were decreased and T cells were most abundant in LC patient-derived PECs. Notably, FCGR3A-expressing macrophages were dominant over DCs and GATA6-expressing macrophages in LC patient-derived PECs. Bulk RNA-seq analysis further clarified expression of a set of genes was up- or down-regulated along with LC severity. Especially, expression of T cell signature genes was featured by its increase at Child-Pugh class B, but decrease at Child-Pugh class C. Flowcytometry analysis showed increase of T cells, decrease of DCs and macrophages, and increased expression of CD16 and CD163 in CD1cintCD14high cells corresponding to FCGR3A-expressing macrophages in LC patient-derived PECs. CONCLUSIONSIn LC patient-derived PECs, myeloid and T cell populations and their gene expression profiles were fluctuated with severity. Our findings should contribute to further development of diagnosis or therapeutic maneuver for LC.

immunology↗

bioRxiv2024

cryoMDM; Molecular simulation Driven structural Matching Approach to Estimate Variety of Atomic Three-dimensional Model Based on Noisy cryoEM Single Particle Images.

Understanding the structure of biomolecules is essential for comprehending life phenomena. In recent years, cryo-electron microscopy (cryoEM) single-particle structure analysis has made remarkable advancements. The single-particle images obtained from cryoEM measurements have a poor signal-to-noise ratio, and existing methods rely on multi-image analysis, which averages multiple measurement data to achieve high-resolution structures. However, this multi-image approach statistically processes several measurement images, causing the unique structural features of individual data to be lost. To overcome this inherent challenge, we developed a molecular simulation-driven structural matching method, cryoMDM, which identifies the plausible structure for cryoEM two-dimensional images from a vast number of three-dimensional (3D) structural model candidates generated through molecular simulations. The innovative aspect of this method is that it enables the estimation of 3D atomic models from a single-particle image, going beyond merely generating a 3D electron density map. By linking each cryoEM particle image to a 3D structure in the simulation space, we can directly connect cryoEM measurement data to continuous structural changes in biomolecules. Applying this method to the spike protein derived from SARS-CoV-2, we successfully captured various intermediate structures of the spike protein, revealing critical information about the early stages of viral entry.

biophysics↗

bioRxiv2024

The structural diversity of telomeres and centromeres across mouse subspecies revealed by complete assemblies

It is over twenty years since the publication of the C57BL/6J mouse reference genome, which has been a key catalyst for understanding mammalian disease biology. However, the mouse reference genome still lacks telomeres and centromeres, contains 281 chromosomal sequence gaps, and only partially represents many biomedically relevant loci. We present the first T2T mouse genomes for two key inbred strains, C57BL/6J and CAST/EiJ. These T2T genomes reveal significant variability in telomere and centromere sizes and structural organisation. We add an additional 213 Mbp of novel sequence to the reference genome containing 517 protein-coding genes. We examined two important but incomplete loci in the mouse genome - the pseudoautosomal region (PAR) on the sex chromosomes and KRAB zinc finger proteins (KZFPs) loci. We identified distant locations of the PAR boundary, different copy number and sizes of segmental duplications, and a multitude of amino acid substitution mutations in PAR genes.

genomics↗

bioRxiv2024

Centralspindlin promotes C. elegans anchor cell specification, vulva induction and morphogenesis

Caenorhabditis elegans vulval development is a relatively simple model of organ development whereby a signal from the overlying gonad induces three epithelial cells to undergo three rounds of cell division to generate 22 cells that make up the vulva. Specification of the vulva cell fates requires coordination between cell division and cell signaling via LIN-12/Notch and LET-23/EGFR pathways in the somatic gonad and the underlying epithelium. Here we characterize the positive regulation of vulval development by the centralspindlin complex, a conserved cytokinesis regulator. Centralspindlin, a heterotetramer of ZEN-4/KIF23 and CYK-4/RacGAP1, is essential for completion of cytokinesis during early embryonic cell divisions. We found that centralspindlin is required in the somatic gonad for division of somatic gonad precursor cells and hence specification of the LIN-3/EGF-secreting anchor cell critical for LET-23/EGFR-mediated vulval induction. However, the requirements for centralspindlin for cytokinesis during postembryonic development are incomplete as a binucleate anchor cell is frequently specified. The presence of the binucleate anchor cell correlates with vulva induction and demonstrates that LIN-12/Notch signaling, required for anchor cell specification, and LET-23/EGFR signaling, required for vulva induction, is largely functional in these cells. Centralspindlin is also partially required for cytokinesis of the vulval cells where it regulates vulva morphogenesis rather than induction. We also found that the GAP domain of CYK-4/RacGAP1 required for contractile ring assembly during embryonic division is not essential for vulval development. Thus, there appears to be different requirements for centralspindlin during postembryonic development of the somatic gonad and vulva as compared to early embryogenesis.

developmental biology↗

bioRxiv2024

Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1

The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPAR{gamma} is mediated by Transportin-1, but PPAR{gamma} lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPAR{gamma} in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPAR{gamma}, indicating that PPAR{gamma} recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPAR{gamma}.

molecular biology↗

bioRxiv2024

Identification and structural characterization of a novel acetyl xylan esterase from Aspergillus oryzae

Acetyl xylan esterase plays a crucial role in the degradation of xylan, the major plant hemicellulose, by liberating acetic acid from the backbone polysaccharides. Acetyl xylan esterase B from Aspergillus oryzae, designated AoAXEB, was biochemically and structurally investigated. The AoAXEB-encoding gene with a native signal peptide was successfully expressed in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 8.0 and 30 {degrees}C, respectively, and was stable up to 35{degrees}C. The optimal substrate for hydrolysis by purified recombinant AoAXEB among a panel of -naphthyl esters was -naphthyl acetate. Recombinant AoAXEB catalyzes the release of acetic acid from wheat arabinoxylan. The release of acetic acid from wheat arabinoxylan increases synergistically with xylanase addition. No activity was detected using the methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. The crystal structures of AoAXEB in the apo and succinate complexes were determined at resolutions of 1.75 and 1.90 [A], respectively. Although AoAXEB has been classified in the Esterase_phb family in the ESTerases and alpha/beta-Hydrolase Enzymes and Relatives (ESTHER) database, its structural features partly resemble those of ferulic acid esterase in the FaeC family. Phylogenetic analysis also indicated that AoAXEB is located between the clades of the two families. Docking analysis provided a plausible binding mode for xylotriose substrates acetylated at the 2- or 3-hydroxy position. This study expands the repertoire of side chain-degrading enzymes required for complete plant biomass degradation.

biochemistry↗

bioRxiv2024

Fc-independent SARS-CoV-2 infection-enhancing antibodies decouple N-terminal and receptor-binding domains by cross-linking neighboring spikes

Antibody dependent enhancement (ADE) is a serious concern in vaccine development. The canonical ADE pathways are dependent on the fragment crystallizable (Fc) region of the antibody. In SARS-CoV-2 several antibodies have been discovered that inflict ADE in vitro. These antibodies target the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. We previously proposed that these NTD-targeting infection-enhancing antibodies (NIEAs) cross-link neighboring spike proteins via their NTDs, and that this results in a decoupling between the NTD and receptor binding domain (RBD), facilitating the "RBD down" to "up" transition. In this study we present a combination of molecular dynamics simulations and cryogenic electron microscopy data that, together, demonstrate that NIEAs are indeed able to cross-link neighboring SARS-CoV-2 spike proteins, and that this cross-linking results in a decoupling of the NTD and RBD domains. These findings provide support for an Fc region independent ADE pathway that is not only relevant for SARS-CoV-2 but also for other viruses of which the spike proteins undergo a conformational change upon host cell entry.

molecular biology↗

bioRxiv2024

Cryo-electron tomography of the actin cytoskeleton and membrane organization during lamellipodia formation using optogenetics

Lamellipodia are sheet-like protrusions essential for migration and endocytosis, yet the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we combined the optogenetic tool PA-Rac1 with cryo-ET to enable ultrastructural analysis of newly formed lamellipodia. We successfully visualized lamellipodia at various extension stages, representing phases of their formation. In minor extensions, several unbundled actin filaments formed "Minor protrusions" at the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed "mini filopodia." In fully extended lamellipodia, filopodia matured at multiple points, and cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This actin turnover likely drives lamellipodial protrusion, providing new insights into actin dynamics and cell migration.

cell biology↗

bioRxiv2024

Tuning of ribosome levels mediated by RNase I and hibernating ribosomes

Protein biosynthesis is an energy-hungry intracellular process that requires the stringent regulation of ribosome abundance under environmental conditions. In response to stress, some active ribosomes are degraded while others, in bacteria, enter a hibernation state to protect against degradation. RNase I, a conserved T2 family ribonuclease in Escherichia coli, degrades ribosomal RNA to suppress biofilm formation, whereas it interacts with ribosomes. However, how and why RNase I binds to ribosomes remains elusive. Here, we show that hibernating ribosomes bind to RNase I and inhibit its activity, thereby promoting biofilm formation. We determined the cryo-electron microscopy structure of the hibernating ribosome complexed with RNase I. RNase I interacts with helix 41 of the 16S rRNA and the ribosomal protein uS14 in the head domain of the 30S subunit of the hibernating ribosome and positions its active centre away from helix 41, resulting in its catalytic inactivation. Hibernating ribosomes are protected from RNase I-mediated cleavage, and our in vivo and in vitro analyses revealed that RNase I targets dissociated large and small ribosomal subunits for rRNA degradation. These findings reveal a previously uncharacterized regulatory strategy that ribosomes modulate RNase I activity, ensuring both the preservation and timely degradation of ribosomes during environmental stress adaptation.

molecular biology↗

bioRxiv2024

ASO-enhancement of TARDBP exitron splicing mitigates TDP-43 proteinopathies

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are fatal neurodegenerative diseases characterized by pathological aggregation and nuclear functional loss of TDP-431,2. Current therapies inadequately address this core pathology3,4, necessitating innovative approaches that target aggregation while preserving TDP-43s essential functions. Here we demonstrate that enhancing the splicing of the TARDBP exitron--a cryptic intron encoding the aggregation-prone intrinsically disordered region (IDR) of TDP-435,6-- effectively mitigates TDP-43 pathology. This exitron splicing event, directly regulated by nuclear TDP-437-9, suppresses the expression of IDR-containing TDP-43 isoforms and generates IDR-spliced-out TDP-43 isoforms7,9,10 (which we term "IDRsTDP"). Our findings reveal that IDRsTDP, known to heterodimerize with full-length TDP-4310, inhibits TDP-43 aggregation by suppressing IDR-mediated clustering and enhances TDP-43 clearance via chaperone-mediated autophagy. In disease states, however, impaired nuclear TDP-43 function disrupts exitron splicing, leading to increased levels of IDR-containing TDP-439,11 and reduced levels of IDRsTDP, exacerbating aggregation and nuclear dysfunction6,12-17. By identifying HNRNPA1 and HNRNPC as key repressors of TARDBP exitron splicing, we designed antisense oligonucleotides (ASOs) to block their binding and restore splicing. These ASOs suppressed TDP-43 pathology and neurodegeneration in both neuronal cell models with impaired nuclear transport and a mouse model of proteasome dysfunction-induced TDP-43 proteinopathy. Our strategy, by rescuing the impaired autoregulatory pathway, inhibits the pathological cycle of TDP-43 aggregation and nuclear dysfunction, offering a promising avenue for treating these currently intractable neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Dual expansion routes likely underlie the present-day population structure in a Parnassius butterfly across the Japanese Archipelago

The Japanese Archipelago, comprising a series of isolated yet interconnected islands, had been geographically separated from the Eurasian continent. The linear topography presents a unique biogeographic context for dispersing organisms from the continent. In this study, we utilized mitochondrial DNA (mtDNA) and single nucleotide polymorphism (SNP) variation to elucidate the dispersal history of the Japanese clouded butterfly Parnassius glacialis across the Japanese Archipelago, including North Island (Hokkaido), Main Island (Honshu) and Shikoku Island. Our analysis of mtDNA (COI, COII) at 1192 base pairs (bps) revealed 49 haplotypes and identified three distinct haplotype groups in the network. These groups correspond geographically to East Japan, West Japan, and Chugoku-Shikoku. The Chugoku-Shikoku group is the most ancient lineage. Interestingly, the Chugoku-Shikoku lineage showed a closer network connection to the East Japan lineage than the geographically proximate West Japan lineage. Divergence time estimates suggest that the Chugoku-Shikoku lineage diverged from the continental P. glacialis approximately 3.05 million years ago (MYA). Subsequently, from the Chugoku-Shikoku lineage, the East Japan and West Japan lineages diverged around 1.05 MYA, with the subsequent divergence between the East and West Japan lineages occurring at approximately 0.62 MYA. Based on the 3067 SNP genotypes, population structure analysis revealed five distinct genetic structures within the Japanese Archipelago, indicating geographical differentiation. From the analyses by mtDNA and SNP variations, four primary genetic barriers were identified: between Hokkaido and Honshu, between East and Central Japan, within the Kansai region, and within the Chugoku region. The former three lines corresponded to the Blakiston Line, the Itoigawa-Shizuoka Tectonic Line, and Lake Biwa, respectively. These findings suggest that P. glacialis diverged from the continental P. glacialis and expanded its range across the Japanese Archipelago via the North and South routes, establishing its current distribution.

molecular biology↗

bioRxiv2024

Structural insight into sodium ion pathway in the bacterial flagellar stator from marine Vibrio

Many bacteria swim in liquid or swarm on surface using the flagellum rotated by a motor driven by specific ion flow. The motor consists of the rotor and stator, and the stator converts the energy of ion flow to mechanical rotation. However, the ion pathway and the mechanism of stator rotation coupled with specific ion flow are still obscure. Here, we determined the structures of the Na+-driven stator of Vibrio, namely PomAB, in the presence and absence of sodium ions and the structure with its specific inhibitor, phenamil, by cryo-electron microscopy. The structures and following functional analysis revealed the sodium ion pathway, the mechanism of ion selectivity, and the inhibition mechanism by phenamil. We propose a model of sodium ion flow coupled with the stator rotation based on the structures. This work provides insights into the molecular mechanisms of ion specificity and conversion of the electrochemical potential into mechanical functions.

microbiology↗