Biology subjects

Kato, Y.

Publications and source records attributed to Kato, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Wsc1 acts as a piezosensor in Saccharomyces cerevisiae, enhancing glycerol efflux via aquaglyceroporin Fps1 in response to high hydrostatic pressure

The fungal cell wall is the initial barrier for the fungi against diverse external stresses, such as osmolarity changes, harmful drugs, and mechanical injuries. This study explores the roles of osmoregulation and the cell wall integrity (CWI) pathway in response to high hydrostatic pressure in the yeast Saccharomyces cerevisiae. We demonstrate the roles of the transmembrane mechanosensor Wsc1 and aquaglyceroporin Fps1 in a general mechanism to maintain cell growth under high-pressure regimes. The promotion of water influx into cells at 25 MPa, as evident by an increase in cell volume and a loss of the plasma membrane eisosome structure, promotes the activation of Wsc1, an activator of the CWI pathway. The downstream mitogen-activated protein kinase Slt2 was hyperphosphorylated at 25 MPa. Glycerol efflux increases via Fps1 phosphorylation, which is initiated by downstream components of the CWI pathway and contributes to the reduction in intracellular osmolarity under high pressure. The elucidation of the mechanisms underlying adaption to high pressure through the well-established CWI pathway could potentially translate to mammalian cells and provide novel insights into cellular mechanosensation.

cell biology↗

bioRxiv2022

A land plant specific VPS13 mediates polarized vesicle trafficking in germinating pollen

Pollen has an extraordinary ability to convert from a dry state to an extremely rapidly growing state. During pollination, pollen receives water and Ca2+ from the contacting pistil, which will be a directional cue for pollen tube germination. The subsequent rapid activation of directional vesicular transport must support the pollen tube growth, but the molecular mechanism leading to this process is largely unknown. Here we show that a plant-specific VPS13, AtVPS13a, mediates vesicle trafficking during the polarization process in Arabidopsis pollen. AtVPS13a knockout severely affected pollen germination and lipid droplet discharge, while Ca2+-dynamics after pollination was unchanged. Cellular distribution patterns of AtVPS13a and a secretory vesicle marker were synchronized, with a slight delay to the Ca2+-dynamics in polarizing pollen. The absence of AtVPS13a led to reduced cell wall deposition during pollen germination. These results suggest that AtVPS13a mediates pollen polarization, by regulating proper directional vesicular transport following Ca2+-signaling.

plant biology↗

bioRxiv2022

A highly contiguous genome assembly of red perilla (Perilla frutescens) domesticated in Japan

Perilla frutescens (Lamiaceae) is an important herbal plant with hundreds of bioactive chemicals, among which perillaldehyde and rosmarinic acid are the two major bioactive compounds in the plant. The leaves of red perilla are used as traditional Kampo medicine or food ingredients. However, the medicinal and nutritional uses of this plant could be improved by enhancing the production of valuable metabolites through the manipulation of key enzymes or regulatory genes using genome editing technology. Here, we generated a high-quality genome assembly of red perilla domesticated in Japan. A near-complete chromosome level assembly of P. frutescens was generated contigs with N50 of 41.5 Mb from PacBio HiFi reads. 99.2% of the assembly was anchored into 20 pseudochromosomes, among which seven pseudochromosomes consisted of one contig, while the rest consisted of less than six contigs. Gene annotation and prediction of the sequences successfully predicted 86,258 gene models, including 76,825 protein-coding genes. Further analysis showed that potential targets of genome editing for the engineering of anthocyanin pathways in P. frutescens are located on the late-stage pathways. Overall, our genome assembly could serve as a valuable reference for selecting target genes for genome editing of P. frutescens.

plant biology↗

bioRxiv2022

Activation of the urotensin-II receptor by anti-COVID-19 drug remdesivir induces cardiomyocyte dysfunction

Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular (CV) side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor (GPCR) screening in combination with structural modeling and found that remdesivir is a selective agonist for urotensin-II receptor (UTS2R). Functionally, remdesivir treatment induced prolonged field potential in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and reduced contractility in neonatal rat cardiomyocytes, both of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants (SNVs) in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related CV events and that genetic variations of UTS2R gene can be a potential risk factor for CV events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future. One Sentence SummaryRemdesivir s activity as a selective agonist of urotensin-II receptor underlies its known cardiotoxicity in anti-viral therapy.

pharmacology and toxicology↗

bioRxiv2022

Identification of a novel chalcone reductase gene for isoliquiritigenin biosynthesis in dahlia (Dahlia variabilis)

Butein is one of flavonoids conferring bright yellow flower color and is a precursor of aurone in some species. Butein is synthesized by two steps, 3-malonyl CoA and 4-coumaloyl CoA are converted to isoliquiritigenin in the first step, and then isoliquiritigenin is converted to butein in the second step. In the first step, chalcone synthase (CHS) and chalcone reductase (CHR) catalyze this reaction, however, CHR has been reported for the isoflavone biosynthesis pathway in legumes, and CHR for butein biosynthesis has not yet been isolated. In this study, we report CHR that is evolutionally different gene from legume species is involved in isoliquiritigenin biosynthesis in dahlia. To isolate CHR gene, we conducted comparative RNA-seq analysis between Shukuhai and its butein-loss lateral mutant Rinka. We found DvCHR showed significant difference in expression levels that encodes an aldo-keto reductase (AKR) 13 family protein, which was phylogenetically different from legume CHRs belonging to AKR4A family. Gene expression levels and genotype of DvCHR were correlated with butein accumulation among various dahlia cultivars. Though single over expression of DvCHR was not able to accumulate isoliquiritigenin in tobacco, co-overexpression of DvCHR with a chalcone glucosyltransferase Am4'CGT and a MYB transcription factor CaMYBA successfully induced isoliquiritigenin accumulation. In addition, DvCHR homologous gene expression was detected from butein or aurone accumulating Coreopsideae species but not from non-butein or non-aurone accumulating Asteraceae species. These results indicated DvCHR functions as chalcone reductase for butein biosynthesis in dahlia, and isoliquiritigenin biosynthesis in Coreopsideae species has been developed independently from legume species.

plant biology↗

bioRxiv2022

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane {beta} sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

biochemistry↗

bioRxiv2021

A machine learning model that emulates experts decision making in vancomycin initial dose planning

Vancomycin is a glycopeptide antibiotic that has been used primarily in the treatment of methicillin-resistant Staphylococcus aureus infections. To enhance its clinical effectiveness and prevent nephrotoxicity, therapeutic drug monitoring (TDM) of trough concentrations is recommended. Initial vancomycin dosing regimens are determined based on patient characteristics such as age, body weight, and renal function, and dosing strategies to achieve therapeutic concentration windows at initial TDM have been extensively studied. Although numerous dosing nomograms for specific populations have been developed, no comprehensive strategy exists for individually tailoring initial dosing regimens; therefore, decision making regarding initial dosing largely depends on each clinicians experience and expertise. In this study, we applied a machine-learning (ML) approach to integrate clinician knowledge into a predictive model for initial vancomycin dosing. A dataset of vancomycin initial dose plans defined by pharmacists experienced in vancomycin TDM (i.e., experts) was used to build the ML model. The target trough concentration was attained at comparable rates with the model- and expert-recommended dosing regimens, suggesting that the ML model successfully incorporated the experts knowledge. The predictive model developed here will contribute to improved decision making for initial vancomycin dosing and early attainment of therapeutic windows.

pharmacology and toxicology↗

bioRxiv2021

C9ORF72 dipeptide repeat proteins disrupt formation of GEM bodies and induce aberrant accumulation of survival of motor neuron protein

A GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. In the neurons of ALS patients, dipeptide repeat proteins (DPRs) are produced from repeat-containing RNAs by an unconventional form of translation, and some of these proteins, especially those containing poly(glycine-arginine) and poly(proline-arginine), are toxic to neurons. Gemini of coiled bodies (GEMs) are nuclear structures that harbor survival of motor neuron (SMN) protein, and SMN is essential for the assembly of U-rich small nuclear ribonucleoproteins (snRNPs) that are central for splicing. We previously reported that GEMs are lost and that snRNP biogenesis is misregulated in the motor neurons of ALS patients. Here we show that DPRs interfere with GEM formation and proper SMN localization in HeLa cells and iPSC-derived motor neurons from an ALS patient with the C9ORF72 mutation. The accumulation of poly(glycine-arginine) markedly reduced the number of GEMs and caused the formation of aberrant cytoplasmic RNA granules that sequestered SMN. These findings indicate the functional impairment of SMN in motor neurons expressing DPRs and may provide a mechanism to explain the vulnerability of motor neurons of C9ORF72-ALS patients.

neuroscience↗

bioRxiv2021

Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.

pharmacology and toxicology↗

bioRxiv2020

Disruption of Z-RNA-binding of ADAR1 induces Aicardi-Goutieres syndrome-like encephalopathy in mice

ADAR1 p150 is an enzyme responsible for adenosine-to-inosine RNA editing. Deletion of ADAR1 p150 results in embryonic lethality with a type I interferon (IFN) signature, caused by aberrant MDA5 sensing unedited transcripts. ADAR1 p150 contains a unique Z-DNA/RNA-binding domain (Z); however, the role of this domain remains unknown. A mutation has been identified in this domain in patients with Aicardi-Goutieres syndrome (AGS), an inherited interferonopathy, suggesting an essential role in avoiding MDA5 activation. Here, we show that a mutation in the Z domain reduces the editing activity of ADAR1 p150 by comparing activity between wild-type and mutated isoforms expressed in Adar1/Adar2 knockout cells. Furthermore, we created Z domain-mutated knock-in mice, which displayed severe growth retardation with abnormal organ development, including AGS-like encephalopathy with a type I IFN signature. These abnormalities were ameliorated by the concurrent deletion of MDA5. Collectively, Z-RNA-recognition contributes to ADAR1 p150-mediated RNA editing, which prevents MDA5 activation.

immunology↗

bioRxiv2020

Immunological memory to SARS-CoV-2 assessed for greater than six months after infection

Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at [≥] 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

immunology↗

bioRxiv2020

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope tag insertion

Antibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We have already demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing a PA12 tag as an inserted epitope. Nevertheless, we also found that the complex formation through the inserted PA12 tag inevitably caused structural change around the insertion site of the target. Hence, we here attempted to improve the insertion method and consequently discovered that utilization of a PA14 tag significantly reduced the structural change in the target. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. Due to this structural property, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Molecular dynamics simulations also indicated that the folding of the target was rigid regardless of the PA14 insertion and the complex formation with the NZ-1 Fab. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.

biophysics↗

bioRxiv2020

Intestinal progenitor P-bodies maintain stem cell identity by suppressing pro-differentiation factors.

The role of Processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. Here, we report that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive Enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4 and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly. Loss of Patr-1-dependent P-bodies leads to a loss of stem cells that is associated with inappropriate translation and expression of EC-fate gene nubbin. Transcriptomic analysis of progenitor cells identifies a cadre of such weakly transcribed pro-differentiation transcripts that are elevated after P-body loss. Altogether, this study identifies a coordinated P-body dependent, translational and transcriptional repression program that maintains a defined set of in vivo stem cells in a state primed for differentiation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/175398v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d57273org.highwire.dtl.DTLVardef@14d057aorg.highwire.dtl.DTLVardef@1a2c4a5org.highwire.dtl.DTLVardef@11c5ba3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDrosophila intestinal progenitor cells contain constitutive and ultrastructurally organized P-bodies. C_LIO_LIA P-body regulator Patr-1 is required for intestinal progenitor cell maintenance. C_LIO_LIEnterocyte (EC) genes such as nubbin are weakly transcribed but not translated in intestinal progenitors. C_LIO_LIP-bodies repress EC gene translation to promote stem cell maintenance. C_LI

cell biology↗

bioRxiv2020

PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade

Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on exhausted T cells during chronic illness. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We reported that the PD-1/PD-L1 pathway is closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Moreover, we found that blocking antibodies targeting PD-1 and PD-L1 restore T-cell functions and may be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway remains unclear for chronic equine diseases, including tumors. In this study, we identified nucleotide sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 monoclonal antibodies (mAbs) against EqPD-L1 using in vitro assays. We also evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a high identity and similarity with homologs from other mammalian species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Importantly, PD-L1 expression was confirmed in EMM tumor tissues by immunohistochemistry. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These results suggest that our anti-PD-L1 mAbs may be useful for investigating the expression and role of the equine PD-1/PD-L1 pathway. Further research is required to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horse.

immunology↗

bioRxiv2019

Alignment of time-course single-cell RNA-seq data with CAPITAL

Recent techniques on single-cell RNA sequencing have boosted transcriptome-wide observation of gene expression dynamics of time-course data at a single-cell scale. Typical examples of such analysis include inference of a pseudotime cell trajectory, and comparison of pseudotime trajectories between different experimental conditions will tell us how feature genes regulate a dynamic cellular process. Existing methods for comparing pseudotime trajectories, however, force users to select trajectories to be compared because they can deal only with simple linear trajectories, leading to the possibility of making a biased interpretation. Here we present CAPITAL, a method for comparing pseudotime trajectories with tree alignment whereby trajectories including branching can be compared without any knowledge of paths to be compared. Computational tests on time-series public data indicate that CAPITAL can align non-linear pseudotime trajectories and reveal gene expression dynamics.

bioinformatics↗

bioRxiv2019

A comparative analysis among ADAR mutant mice reveals site-specific regulation of RNA editing

Adenosine-to-inosine RNA editing is an essential posttranscriptional modification catalyzed by adenosine deaminase acting on RNA (ADAR)1 and ADAR2 in mammals. For numerous sites in coding sequences (CDS) and microRNAs (miRNAs), editing is highly conserved and has significant biological consequences, for example, by altering amino acid residues and target recognition. However, technical limitations have prevented a comprehensive and quantitative study to determine how specific ADARs contribute to each site. Here, we developed a simple method in which each RNA region with an editing site was amplified separately and combined for deep sequencing. Using this method, we compared the editing ratios of all sites that were either definitely or possibly conserved in CDS and miRNAs in the cerebral cortex and spleen of wild-type mice, Adar1E861A/E861AIfih-/- mice expressing inactive ADAR1 (Adar1 KI) and Adar2-/-Gria2R/R (Adar2 KO) mice. We found that the editing ratio was frequently upregulated in either Adar mutant mouse strain. In contrast, we found that the presence of both ADAR1 and ADAR2 was required for the efficient editing of specific sites. In addition, some sites, such as miR-3099-3p, showed no preference for either ADAR. We further created double mutant Adar1 KI Adar2 KO mice and observed viable and fertile animals with complete absence of editing, suggesting that ADAR1 and ADAR2 are the sole enzymes responsible for all editing sites in vivo. Collectively, these findings indicate that editing is regulated in a site-specific manner by the different interplay between ADAR1 and ADAR2.

molecular biology↗

bioRxiv2019

Divisively normalized integration of multisensory error information develops motor memories specific to vision and proprioception

Both visual and proprioceptive information contribute to accurate limb movement, but the mechanism of integration of these different modality signals for movement control and learning remains controversial. Here, we aimed to elucidate the mechanism of integration by examining motor adaptation when various combinations of visual and proprioceptive perturbations were applied during reaching movements. We found that the movement corrections by adaptation were explained by a mechanism known as a divisive normalization, which was previously reported to be the mechanism underlying the integration of multisensory signals in neurons. Furthermore, we found evidence that the motor memory for each sensory modality was formed separately and the outputs from these memories were integrated. These results provide a novel view of the utilization of different sensory modality signals in motor control and adaptation.

neuroscience↗

bioRxiv2019

CDK1 dependent phosphorylation of hTERT contributes to cancer progression

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses revealed that phosphorylation of hTERT at threonine 249 occurs more frequently in advanced cancers. Using CRISPR/Cas9 genome editing, we introduced substitution mutations at threonine 249 in the endogenous hTERT locus and found that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase activity. Cap Analysis of Gene Expression (CAGE) demonstrated that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomerase independent manner.

cancer biology↗