Biology subjects

Nagata, S.

Publications and source records attributed to Nagata, S..

16 recordsLinked to original sources

bioRxiv2026

A versatile and stable reporter platform for human rhinovirus and other enteroviruses

Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.

microbiology↗

bioRxiv2026

A stepwise model for asp gene emergence across HIV and SIV lineages: progressive stop-codon loss and regulatory evolution

Background The antisense protein (ASP), encoded by an open reading frame (ORF) overlapping the env gene in human immunodeficiency virus (HIV), was proposed over 30 years ago. ASP is an ~189-amino-acid-long, highly hydrophobic protein with proposed roles in autophagy and viral entry. Although primarily described in pandemic HIV-1 group M, ASP was also identified in a single simian immunodeficiency virus (SIVcpz) isolate, suggesting recent acquisition. However, SIV from more distantly related lineages, including Old World monkeys (OWM), remains largely unexamined, and the broader origin of ASP is unresolved. Results We conducted computational analyses of 91,245 HIV-1, HIV-2, and SIV env sequences to assess the coding potential and evolutionary history of asp across primate lentiviruses. The sequences were classified into seven types based on the asp-frame ORF length and continuity: ASP, S1, and S2 for single continuous ORFs ([≥] 450, 300-449, and 120-299 nucleotides, respectively); F1-F3 for fragmented ORFs of comparable combined length; and Z for sequences lacking a detectable ORF of <120 nucleotides. Many SIV sequences retained a single, uninterrupted ORF of <120 nucleotides in the ASP frame, comparable to HIV-1 ASP-type sequences. A phylogenetic tree of 599 env sequences divided strains into HIV-1/SIVcpz/SIVgor and more distant HIV-2/SIV lineages. Fragmented asp ORFs were concentrated among basal, distantly related strains, suggesting asp emerged from stop-codon-rich ancestral segments. The stop-codon density progressively declined with ORF continuity in both groups ({rho} = -0.63 and -0.77, respectively; both p < 0.001), consistent with the stop-codon-loss mechanism proposed previously for HIV-1 and extended to SIV here. The nonsynonymous/synonymous analysis revealed strong purifying selection within the overlapping region in HIV-1, and a more variable, lineage-dependent pattern in SIV. Motif analysis of the U3/LTR-like regions showed antisense promoter-associated transcription factor binding sites, including NF-{kappa}B and ETS1, were enriched in HIV-1, SIVcpz, and SIVgor. Conclusions These findings support a stepwise model of antisense regulatory evolution, whereby promoter-associated transcription factor motifs accumulated before a continuous asp ORF became fixed in HIV-1. Variation within the pandemic lineage suggests that the asp coding potential remains evolutionarily dynamic, providing new insights into the emergence and stabilization of overlapping genes during lentiviral evolution.

evolutionary biology↗

bioRxiv2026

From neuropeptide and receptor annotation to ligand-receptor pairing: a sequence- and structure-based framework for mapping the neuropeptide-receptor interactome in Gryllus bimaculatus

Neuropeptides and their G protein-coupled receptors (GPCRs) control much of insect physiology and behaviour, but in Gryllus bimaculatus, an emerging model and edible insect, receptor sequence similarity hinders the mapping of which peptide each GPCR activates. We re-annotated a chromosome-scale genome (BUSCO 95.3%, from 86.7% on insecta_odb12) with comprehensive curation of 48 neuropeptide precursor families (51 loci, including seven not previously identified) and 134 candidate GPCRs (66 rhodopsin-class, 68 secretin-class), providing a near complete neuropeptide-receptor interactome catalogue. We modelled all 15,946 peptide-receptor pairs with AlphaFold3 and Boltz-2 and scored each interface with pLDDT and ipSAE. Ranking these scores, and cross-checking the top candidate for each family against a receptor phylogeny of known ligand specificity, gave a confident, phylogenetically related receptor for 27 of 35 curated receptor groups. These matches confirm the structural scorings with existing deorphanization data and propose receptors for peptides with no prior functional evidence. The annotation, curated peptide and receptor sets, and ranked complexes are available through CricketBase (https://cricket.annotation.jp), a genome browser with a structure viewer of peptide-receptor complexes, providing a resource for G. bimaculatus endocrinology and a workflow to deorphanize GPCRs in other non-model insects.

bioinformatics↗

bioRxiv2026

Identification of tail-binding proteins of Arabidopsis class VIII myosin ATM1 using TurboID proximity labeling and AlphaFold3

Higher plants possess two classes of myosin molecular motors, class XI and class VIII, both unique to the plant lineage. The diverse cellular functions of class XI myosins, including organelle transport and nuclear positioning, have been elucidated largely through systematic identification of cargo adaptor proteins that bind to their globular tail domains (GTDs). In contrast, no proteome-wide screen for class VIII myosin tail-binding proteins has been reported; the few known interacting proteins were each discovered through studies focused on the binding partner rather than on the myosin itself, leaving the full repertoire of class VIII myosin-associated proteins largely unknown. Here, we employed TurboID-based proximity labeling to systematically identify proteins associated with the GTD of the class VIII myosin ATM1 in Arabidopsis thaliana, as this approach covalently biotinylates neighboring proteins in vivo, enabling their identification even after proteolytic degradation during cell lysis. We identified 233 non-redundant candidate ATM1-proximal proteins. Candidates were prioritized by AlphaFold3-based protein complex structure prediction and validated by co-immunoprecipitation. We identified two ATM1-associated proteins: C3H61/AtTZF5, a tandem zinc finger protein involved in mRNA turnover at processing bodies and stress granules; and SFH7, a Sec14-nodulin domain protein that mediates phosphatidic acid transfer from the endoplasmic reticulum to chloroplasts. These findings provide initial evidence linking ATM1 to proteins involved in post-transcriptional gene regulation and interorganellar lipid transport, raising the possibility of previously unrecognized connections between class VIII myosins and these cellular processes.

plant biology↗

bioRxiv2025

A T-cell Intrinsic Role for APOL1 Risk Alleles in Allograft Rejection

African Americans have an increased risk of kidney disease due to exonic variants in Apolipoprotein-L1 (G1 and G2). These prevalent variants have also been linked with kidney rejection, but outside of association with African ancestry, underpinning causal mechanisms are unknown. We investigated T-cell function using transgenic mice with physiologic expression of wild type (G0-), G1-, or G2-APOL1. Mice with variant APOL1 showed greater CD8+T-cell activation with expansion of a central memory (TCM) subset. Stimulated G1-CD8+T-cells showed enhanced proliferation and cytokine production, which reversed with APOL1 inhibition. In MHC-mismatched cardiac transplants, G1-mice demonstrated greater CD8+T-cell infiltration and reduced survival. Bulk transcriptome of G1-CD8+T-cells, and single-cell transcriptome of graft infiltrating TCMs, showed enrichment of canonical T-cell receptor (TCR) pathways including Ca2+-signaling. G1-CD8+T-cells demonstrated baseline ER-Ca2+ depletion followed by sustained increases in cytosolic-Ca2+ upon TCR stimulation. G1-CD8+T-cells were more sensitive to Ca2+ chelation, or store-operated Ca2+ entry inhibition, and relatively resistant to calcineurin antagonism vs. G0-CD8+T-cells. Analogously, in a kidney transplant cohort, APOL1-variant recipients developed rejection when they had elevated peripheral TCMs before transplantation and despite significantly higher tacrolimus levels vs G0/G0-AAs with rejection. In summary, we unravel an excitatory T-cell intrinsic mechanism for APOL1 exonic variants, causally linking them with kidney rejection.

immunology↗

bioRxiv2024

Two neuropeptide signalings regulate post-mating refusal behavior and reproductive system in male crickets

After mating, insects always perform mating refusal behavior, termed post-mating refractoriness, due to physiological restrictions. Male crickets, Gryllus bimaculatus, characteristically exhibit 1-hour post-mating refractory stage, controlled by terminal abdominal ganglion. The molecular mechanisms underlying the male-specific precisely timed refractory stage remain elucidated. Here we show that among 28 neuropeptide precursors expressed in the terminal abdominal ganglion, DH31, myosuppressin, allatotropin, and sNPF exhibited male-specific expression based on RT-qPCR and in situ hybridization. However, RNA interference experiments showed that only knockdown of allatotropin and sNPF changed the duration of the refractoriness. Furthermore, allatotropin and sNPF knockdown influenced functions of male reproductive system by inhibiting seminal fluid secretion from male accessory gland and decreasing sperm storage in seminal vesicles, respectively. Knockdown of their receptors caused similar phenotypes. In conclusion, this study demonstrated the regulation of post-mating refusal behavior and reproductive system by Allatotropin and sNPF signalings in male crickets.

physiology↗

bioRxiv2024

Generation of antagonistic biparatopic anti-CD30 antibody from an agonistic antibody by precise epitope determination and utilization of structural characteristics of CD30 molecule

CD30 is a type I membrane protein that has been successfully targeted for lymphoma therapy using Brentuximab vedotin, an antibody-drug conjugate. Recently, the potential of blocking CD30-dependent NF-{kappa}B intracellular signaling has gained attention for treating inflammatory disorders. Development of antibody-based CD30 antagonists would broaden therapeutic strategies. A challenge in developing antagonistic antibodies is that the bivalent form of natural antibody format inevitably cross-links trace amounts of CD30 molecules, leading to signal transduction. In this study, we developed a series of biparatopic antibodies with each pair of antibody variable domains (Fvs) binding to distinct epitopes on CD30, and evaluated their biological activities and binding modes. Initially, we precisely identified epitope sites of the nine antibodies precisely by assessing binding to multiple orthologous CD30 proteins and mutants. We then produced 36 biparatopic antibodies covering all possible combinations of the nine Fvs, and analyzed their biological activities. Among these, we identified both potent agonists and antagonists. Notably, a significant proportion of the biparatopic antibodies displayed reduced agonistic activities, including 1:1-binding antagonists derived from a Fv of a strong agonist previously developed for lymphoma therapy, AC10. The mechanism of signaling activity induction is discussed using epitope information, which leads to the strategies of the development of biparatopic antibodies.

biochemistry↗

bioRxiv2024

Discovery of anti-SARS-CoV-2 S2 protein antibody CV804 with broad-spectrum reactivity with various beta coronaviruses and analysis of its pharmacological properties in vitro and in vivo

SARS-CoV-2 pandemic alerts us that spillovers of various animal coronaviruses to human in the future may bring us enormous damages. Thus, there is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses.CV804 against the S2 domain of the spike protein, which is less prone to mutations. CV804 shows not only broad cross-reactivities with representative 20 animal-origin coronaviruses but also with diseases-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and mutant strains of SARS-CoV-2. Other than that, the main characteristics of CV804 are that it has strong antibody-dependent cellular cytotoxicity (ADCC) activity to SARS-CoV2 spike protein-expressed cells in vitro and completely lacks virus-neutralization activity. Comprehensively in animal models, CV804 suppressed disease progression by SARS-CoV-2 infection. Structural studies using HDX-MS and point mutations of recombinant spike proteins revealed that CV804 binds to a unique epitope within the highly conserved S2 domain of the spike proteins of various coronaviruses. Based on the overall data, we suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein expressed on the surface of the infected cells and weakens the viral virulence by supporting host immune cells attack through ADCC activity in vivo. CV804 epitope identified in this study is not only useful for the design of pan-corona antibody therapeutics but also to design next-generation coronavirus vaccines and antiviral drugs.

microbiology↗

bioRxiv2022

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by epitope selection

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we developed BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulated the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. One particular antagonist BpAb bound TNFR2 in 1:1 ratio without unwanted signal transduction, with its structural basis revealed by cryo-electron microscopy. This antagonist suppressed the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

biochemistry↗

bioRxiv2022

Two gustatory receptors involved in host plant recognition of silkworm larvae

Herbivorous insects can identify their host plants by sensing plant secondary metabolites as chemical cues. We previously reported the two-factor host acceptance system of the silkworm Bombyx mori larvae. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercitrin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. Nevertheless, the molecular basis for the ultrasensitive sensing of these compounds remains unknown. In this study, we demonstrated that two gustatory receptors (Grs), BmGr6 and BmGr9, are responsible for sensing the mulberry compounds with attomolar sensitivity for host plant recognition by silkworm larvae. Calcium imaging assay using cultured cells expressing the silkworm putative sugar receptors (BmGr4-10) revealed that BmGr6 and BmGr9 serve as receptors for CGA and ISQ with attomolar sensitivity in human embryonic kidney 293T cells. CRISPR/Cas9-mediated knockout (KO) of BmGr6 and BmGr9 resulted in a low probability of making a test bite of the mulberry leaves, suggesting that they lost the ability to recognize host leaves specifically. Electrophysiological recordings showed that the loss of host recognition ability in the Gr-KO strains was due to a drastic decrease in MP sensitivity toward ISQ in BmGr6-KO larvae and toward CGA and ISQ in BmGr9-KO larvae. Our findings unraveled that the two Grs, which have been regarded as sugar receptors, are molecules responsible for detecting plant phenolics in host plant recognition.

neuroscience↗

bioRxiv2022

Impact of reinfection with SARS-CoV-2 Omicron variants in previously infected hamsters

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

microbiology↗

bioRxiv2021

Distinct Expansion of Group II Introns Depends on the Type of Intron-encoded Protein and Genomic Signatures in Prokaryotes

Group II introns (G2Is) are self-splicing ribozymes that have retroelement characteristics in prokaryotes. Although G2Is are considered an important factor in the evolution of prokaryotes, comprehensive analyses of these introns among the tens of thousands of prokaryotic genomes currently available are still limited. Here, we developed a bioinformatic pipeline that systematically collects G2Is and applied it to prokaryotic genomes. We found that in bacteria, 25% (447 of 1,790) of the total representative species had an average of 5.3 G2Is, and in archaea, 9% (28 of 296) of the total representative species had an average of 3.0 G2Is. The greatest number of G2Is per species was 101 in Arthrospira platensis (phylum Cyanobacteriota). A comprehensive sequence analysis of the intron-encoded protein (IEP) in each G2I sequence was conducted and resulted in the addition of three new IEP classes (U1-U3) to the previous classification. This analysis suggested that about 30% of all IEPs are noncanonical IEPs. The number of G2Is per species was defined almost at the phylum level, and the type of IEP was associated as a factor in the G2I increase, i.e. there was an explosive increase in G2Is with bacterial C-type IEPs in the phylum Firmicutes and in G2Is with CL-type IEPs in the phylum Cyanobacteriota. We also systematically analyzed the relationship between genomic signatures and the mechanism of these increases in G2Is. This is the first study to systematically characterize G2Is in the prokaryotic phylogenies.

evolutionary biology↗

bioRxiv2021

An intramolecular scrambling path controlled by a gatekeeper in Xkr8 phospholipid scramblase

Xkr8-Basigin is a phospholipid scramblase at plasma membranes that is activated by kinase or caspase. We investigated its structure at a resolution of 3.8[A]. Its membrane-spanning region had a cuboid-like structure stabilized by salt bridges between hydrophilic residues in helices in the lipid layer. The molecule carried phosphatidylcholine in a cleft on the surface that may function as an entry site for phospholipids. Five charged residues placed from top to bottom inside the molecule were essential for providing a path for scrambling phospholipids. A tryptophan residue was present at the extracellular end of the pathway and its mutation made the Xkr8-Basigin complex constitutively active, indicating its function as a gatekeeper. The structure of Xkr8-Basigin provides novel insights into the molecular mechanisms underlying phospholipid scrambling.

biochemistry↗

bioRxiv2020

Human iPSC-derived hypertrophic chondrocytes reveal a mutation-specific unfolded protein response in chondrodysplasias

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, utilizing human chondrodysplasia-specific iPSCs, we examined the UPR caused by mutations in MATN3 or COL10A1. In growth plate-like structures formed from iPSC-derived sclerotome in vivo, the hypertrophic zone was disrupted, and induced hypertrophic chondrocytes in vitro showed varying levels of ER stress depending on the mutation. Autophagy inducers and chemical chaperones succeeded in reducing ER stress only in some mutants, while transcriptome analysis revealed many mutation-specific changes in genes involved in apoptosis, metabolism, and protein trafficking. In this way, our system has allowed the precise evaluation of the UPR caused by each mutation, opening up new avenues for treatment of individual chondrodysplasia patients.

cell biology↗

bioRxiv2019

Crystal structure of a human plasma membrane phospholipid flippase

ATP11C, a member of P4-ATPase flippase, exclusively translocates phosphatidylserine from the outer to the inner leaflets of the plasma membrane, and maintains the asymmetric distribution of phosphatidylserine in the living cell. However, the mechanisms by which ATP11C translocates phosphatidylserine remain elusive. Here we show the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in an outward-open E2P conformation. Two phosphatidylserine molecules are in a conduit that continues from the cell surface to the occlusion site in the middle of the membrane. Mutations in either of the phosphotidylserine binding sites or along the pathway between significantly impairs specific ATPase and transport activities. We propose a model for phosphatidylserine translocation from the outer to the inner leaflet of the plasma membrane.

biochemistry↗

bioRxiv2019

The two-step chemosensory system underlying the oligophagy of silkworm larvae

Oligophagous insect herbivores specifically identify host-plant leaves using their keen sense of taste1. Plant secondary metabolites and sugars are key chemical cues for insects to identify host plants and evaluate their nutritional value, respectively2. However, it is poorly understood how the insect chemosensory system integrates the information from various gustatory inputs. Here we report that a two-step chemosensory system is responsible for host acceptance by larvae of the silkworm Bombyx mori, a specialist for several mulberry species. The first step controlled by a gustatory organ, the maxillary palp (MP), is host-plant recognition during palpation at the leaf edge. Surprisingly, MP detects chlorogenic acid, quercetin glycosides, and {beta}-sitosterol, which stimulate feeding by the silkworm3-6, with ultra-sensitivity (thresholds of aM to fM). Detecting a mixture of these compounds triggers test biting. The second step is evaluation of the sugar content in the leaf sap exuded by test biting. Low-sensitivity chemosensory neurons in another gustatory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting. Our present work shows the behavioral and neuronal basis of host acceptance in the silkworm, mainly driven by six phytochemicals. It also reveals that the ultra-sensitive gustation via MP strictly limits initiation of feeding in the silkworm unless it detects a certain combination of host compounds, suggesting an essential role of MP in host-plant selection. The two-step chemosensory system reported herein may commonly underlie stereotyped feeding behavior in phytophagous insects and determine their feeding habits.

ecology↗