Biology subjects

Ito, N.

Publications and source records attributed to Ito, N..

At least 19 recordsLinked to original sources

bioRxiv2026

RISOP, a Reference-Assisted Approach for Enhanced Identification of Oxidized Phospholipids

Oxidized phospholipids (OxPLs) play critical roles in inflammation, ferroptosis, and other oxidative stress-associated processes, yet their systematic characterization in biological systems remains a major analytical challenge owing to their low abundance and vast structural diversity. Here we report RISOP (Reference-Assisted Identification of Sample-specific Oxidized Phospholipids), an untargeted LC-MS/MS workflow that leverages chemically enriched OxPL reference pools to substantially improve OxPL annotation. Reference pools encompassing diverse oxidative modifications and a wide abundance range were experimentally generated using Fenton reaction and H2O2 treatment, providing broad coverage of OxPLs. Reference-assisted integrative analysis yielded a more than twofold increase in identified OxPL species in biological samples under elevated oxidative stress, as demonstrated in ML210-treated cells, a model of ferroptotic stress. We further show that the widely used BODIPY C11 lipid peroxidation probe captures cellular oxidative burden in only a subset of OxPLs identified by RISOP, highlighting the importance of untargeted, comprehensive OxPL profiling. Overall, RISOP provides a versatile platform readily applicable to other classes of oxidized complex lipids for comprehensive characterization in physiological and pathological contexts. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/728773v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@482111org.highwire.dtl.DTLVardef@173126corg.highwire.dtl.DTLVardef@1be7238org.highwire.dtl.DTLVardef@3cf7f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

Short-chain fatty acids modulate the development and the cell surface molecule expression of dendritic cells by epigenetic regulation

BackgroundShort-chain fatty acids (SCFAs) are produced by the gut microbiota as secondary metabolites during fermentation process of dietary fibers. Although SCFAs are beneficial for immuno-related diseases because they regulate the gene expression and functions of myeloid cells, the effects of SCFAs on the development of DCs remain unclear. MethodsWe analyzed the effect of SCFAs on the expression levels of surface proteins and mRNAs, and histone modification in Flt3L-induced bone marrow-derived DCs. ResultsSCFAs, particularly butyrate, regulated the expression of surface molecules on mouse bone marrow-derived dendritic cells (DCs): increases in MHCII, CD86, CD11b, and LPAM-1 (4{beta}7) levels and the ratio of CD11c+/PDCA-1-/B220- conventional DCs (cDCs) to CD11c+/PDCA-1+/B220+ plasmacytoid DCs (pDCs). Experiments using inhibitors of histone deacetylase (HDAC) and Gi proteins, and GPR109A deficient mice indicated that butyrate regulated DCs by suppression of HDACs and not through a stimulatory effect on G protein-coupled receptors. Butyrate and the HDAC inhibitor, trichostatin A (TSA), increased the cDC/pDC ratio, surface LPAM-1 and Itga4 mRNA, while the mRNA level of Itgb7 was not affected by butyrate and was reduced by TSA. ChIP assays showed that butyrate and TSA increased histone acetylation in the Itga4 and Spi1 genes. Furthermore, the butyrate treatment increased the levels of Spi1 mRNA and PU.1 protein and decreased those of Spib/SpiB in DCs. In knockdown (KD) experiments using siRNAs, the gene expression of Itga4 was decreased by KD of Spi1 or Irf8, and cDC/pDC ratio decreased by Spi1 KD. ConclusionsButyrate controls the gene expression and development of DCs through epigenetic regulation and DC-related transcription factors.

immunology↗

bioRxiv2025

Application of the CPER reverse genetics system for genetic engineering of rabies virus

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for E. coli transformation and cloning of RABV full genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.

microbiology↗

bioRxiv2025

Intrinsically Dominant Conformational Diversity in PDZ1 within the Tandem PDZ1-PDZ2 of Human Syntenin-1 Underlined by Crystal Structures.

The intrinsic dynamic asymmetry between homologous PDZ domains in multidomain scaffold proteins offers critical insights into evolutionary mechanisms enabling multivalent partner recognition. Through systematic X-ray crystallographic analysis of human syntenin-1s PDZ1-PDZ2 tandem, we resolve nine high-resolution structures that uncover fundamental differences in conformational plasticity between these sequentially similar domains. Pairwise root-mean-squared deviation (RMSD) analysis of 20 PDZ1 structures across multiple crystal forms reveals substantial structural variability concentrated in the Lys119-Ile125 and Ala181-Glu184 loops - key regions governing ligand specificity within PDZ1s binding cleft. In stark contrast, PDZ2 maintains remarkable structural conservation across all crystallographic environments, indicating divergent evolutionary constraints on these tandem domains. Crucially, comparative analysis of isotropic B-factors demonstrates their inadequacy in capturing the full scope of conformational heterogeneity, emphasizing the necessity of multi-structure comparisons for mapping dynamic landscapes. Molecular dynamics (MD) simulations implemented through GROMACS corroborate these crystallographic observations, showing elevated residue-specific fluctuation (RMSF) values in PDZ1s ligand-binding interface compared to analogous PDZ2 regions. This consistency across experimental and computational approaches confirms that PDZ1s conformational diversity represents an inherent biophysical property rather than crystallographic artifact. The observed dynamic asymmetry suggests a functional division of labor: PDZ1s structural plasticity enables broad ligand recognition via conformational selection mechanisms, while PDZ2s rigid architecture likely stabilizes the tandem domain arrangement. These findings provide an atomic-level rationale for syntenin-1s pleiotropic roles in cellular signaling and establish a structural blueprint for developing domain-selective therapeutics. Given syntenin-1s clinical relevance in cancer metastasis, viral pathogenesis, and neurodevelopmental disorders, our work advances strategies for selectively modulating PDZ1-mediated interactions while preserving PDZ2s scaffolding functions through structure-guided inhibitor design. Highlights{lozenge} Crystal structures of human Syntenin-1s PDZ1-PDZ2 tandem reveal intrinsic conformational plasticity in PDZ1, particularly in ligand-binding loops, contrasting with PDZ2s rigid architecture {lozenge}Pairwise RMSD analysis of 20 PDZ1 structures demonstrates substantial structural variability in the Lys119-Ile125 and Ala181-Glu184 loops, key regions governing ligand specificity {lozenge}Molecular dynamics simulations confirm that PDZ1s conformational diversity is an inherent biophysical property, not a crystallographic artifact {lozenge}The asymmetric dynamics between PDZ1 and PDZ2 suggest a functional division: PDZ1s plasticity enables broad ligand recognition while PDZ2 stabilizes the tandem arrangement {lozenge}These findings provide a structural basis for developing domain-selective Syntenin-1 inhibitors with potential applications in cancer metastasis, viral pathogenesis, and neurodevelopmental disorders

biophysics↗

bioRxiv2025

The role of GATA2 in the expression of the soluble decoy receptor ST2/IL1RL1 in human and mouse mast cells

The ST2/IL1RL1 gene encodes a receptor subunit for IL-33. The ST2/IL1RL1 gene is transcribed and translated to two splice variants, full-length ST2 (ST2L) and soluble ST2 (sST2). ST2L is expressed on cell surface as membrane bound molecule and forms a heterodimer with IL-1RAcP, which plays an important role in Th2 transduces a signaling of IL-33. In contrast, sST2 blocks the IL-33-signaling via functioning as a decoy receptor of IL-33. In this study, we analyzed the regulatory mechanisms of the gene expression of sST2 in mast cell, the major source of sST2. We found that a hematopoietic cell-specific transcription factor GATA2 is essential for sST2 expression in human mast cells (a cell line and primary cells) and mouse mast cells. Various assays including ChIP assay, reporter assay, and 3C assay revealed that GATA2 binding to the distal promoter of the IL1RL1 gene transcriptionally activates proximal promoter-driven expression of sST2 through chromosomal conformation.

immunology↗

bioRxiv2025

PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes

Mucosal mast cells (MMCs) are distinguished from connective tissue MCs by the specific expression of integrin CD103 (E/{beta}7) and MC proteases Mcpt1 and Mcpt2. Although the expression of the Mcpt1 and Mcpt2 genes is cooperatively regulated by the transcription factor GATA2 and TGF-{beta} signaling in MMCs, the transcriptional mechanism of CD103 expression remains unknown. Here, we found that surface CD103 and Itgae mRNA levels were significantly increased by the knockdown (KD) of Gata2 in bone marrow-derived MCs (BMMCs), which was accelerated by a TGF-{beta} stimulation. Since the mRNA levels of Spi1 (encoding PU.1) were increased in Gata2 KD BMMCs, we examined the effects of PU.1 on CD103 expression. As expected, CD103 levels on BMMCs were significantly decreased by Spi1 KD and increased by Spi1 overexpression. Spi1 KD suppressed Itgae expression even in the presence of TGF-{beta} in BMMCs and peritoneal MCs, whereas Gata2 KD amplified the TGF-{beta}-induced increase in Itgae expression. The amount of PU.1 binding to the cis-element in the Itgae gene was significantly and moderately increased by Gata2 KD and the TGF-{beta} stimulation, respectively. Since PU.1 is an essential transcription factor for dendritic cells (DCs), we examined the role of PU.1 in CD103 expression on DCs. The KD experiment using BMDCs showed a significant decrease in CD103 levels in Spi1 siRNA-transfected BMDCs. We concluded that PU.1 affected CD103 expression on MMCs and DCs by transactivating the Itgae gene, and also that GATA2, which positively regulated the MMC-specific expression of Mcpt1 and 2, inhibited CD103 expression by repressing PU.1.

molecular biology↗

bioRxiv2024

Circulating extracellular microRNAs in the blood promote sociability in mice

Extracellular vesicles (EVs) are cell-derived small membrane vesicles and circulate throughout the body, but the impact of circulating EVs on brain function and behavior remains elusive. Here, we report that wild-type (WT) mouse blood, particularly EVs, increases sociability in socially impaired immunodeficient Rag1-/- mice, mimicking the effects of WT T cell transfer. These EVs localized to neurons and regulated PKC{varepsilon} expression, GABAA receptor synaptic localization, and inhibitory postsynaptic signaling in prefrontal cortex (PFC) pyramidal neurons. Injection of Rag1-/- EVs supplemented with miR-23a-3p and miR-103-3p enhanced synaptic function and sociability in Rag1-/- mice. T cells secreted miR-23a-3p via EVs, and Mir23a-/- T cells failed to increase sociability. Similar beneficial effects of WT blood EVs were observed in additional mouse models with sociability deficits, Cntnap2-/- and Shank3-/- mice. These findings uncover a previously unrecognized role of EV miRNAs in mediating immune modulation of synaptic function and social behavior, revealing a novel molecular pathway for immune-neuron communication.

neuroscience↗

bioRxiv2024

Roles of C/EBPα, GATA2, TGF-β-signaling, and epigenetic regulation in the expression of basophil-specific protease genes

In the present study, we analyzed the transcriptional regulation of genes encoding basophil-specific proteases Mcpt8 and Mcpt11 to clarify the molecular mechanisms by which the commitment between basophil and mast cell (MC) is determined. We used bone marrow-derived (BM) cells maintained in the presence of IL-3, in which basophil-like cells and MC-like cells exist. Knock down (KD) and overexpression of a transcription factor C/EBP showed that Cebpa mRNA levels were associated with those of Mcpt8 and Prss34 (a gene symbol of Mcpt11). Treatment with trichostatin A (TSA), a histone deacetylase inhibitor, decreased mRNA levels of Cebpa, Mcpt8, and Prss34 in BM cells, whereas these mRNA levels were increased by the treatment with an inhibitor of DNA methyltransferase. TSA treatment upregulated mRNA levels of Gata1 and Mitf in BM cells, and Mitf KD but not Gata1 KD increased mRNA levels of Cebpa and Prss34. Furthermore, Gata2 KD significantly decreased mRNA levels of Mcpt8 and Prss34 without affecting Cebpa mRNA level. In addition, mRNA levels of Gata1 and Mitf were increased in BM cells transfected with Cebpa siRNA, and decreased in Cebpa-overexpressing BM cells. We also found the TGF-{beta} treatment increased mRNA levels of Mcpt8, Prss34, along with upregulation of Gata2 and Cebpa transcription. Taken together, we conclude that the transcription of basophil-specific protease genes was positively regulated by C/EBP, GATA2, and TGF-{beta} signaling with modification of epigenetic regulation.

immunology↗

bioRxiv2024

Kaempferol exerts anti-inflammatory effects by accelerating Treg development via AhR-mediated and PU.1/IRF4-dependent transactivation of the Aldh1a2/RALDH2 gene in dendritic cells

Retinaldehyde dehydrogenase 2 encoded in the Aldh1a2 gene (RALDH2) is expressed in intestinal dendritic cells (DCs) in mesenteric lymph nodes (MLNs) and plays a crucial role in Treg development by producing retinoic acid. In the present study, we screened food ingredients and identified kaempferol, a flavonoid, as the compound that most effectively up-regulated RALDH2 expression in DCs. The development of Foxp3+ cells from OT-II-derived naive CD4+ T cells was enhanced by a co-culture with OVA peptide-pulsed bone marrow-derived DCs (BMDCs) in the presence of kaempferol or by a pre-treatment of BMDCs with kaempferol. The involvement of the aryl hydrocarbon receptor (Ahr) in the effects of kaempferol on Aldh1a2 expression was demonstrated by in vitro experiments using siRNA and various agonistic or antagonistic compounds of AhR. AhR suppressed Aldh1a2 gene expression and kaempferol inhibited the AhR activity by functioning as an antagonist of AhR. The kaempferol treatment also increased the protein levels of PU.1 and IRF4, which are essential for transcription of the Aldh1a2 gene in DCs, and accelerated the subsequent recruitment of PU.1 to the Aldh1a2 gene. The kaempferol-induced increase in PU.1 protein levels occurred in an AhR-dependent and transcription-independent manner, whereas the kaempferol treatment transactivated the Irf4 gene in DCs. The frequency of DCs exhibiting RALDH2 activity in the MLNs of mice was increased by the intraperitoneally (i.p.) administration of an AhR antagonist or by the oral administration of astragalin, a 3-O-glucoside of kaempferol. We also observed an increase in Tregs in the Peyers patches of C57BL/6 mice i.p. administered kaempferol-treated BMDCs. We utilized an OVA-induced food allergy model of Balb/c mice to examine the effects of kaempferol in vivo and confirmed that the rapid decrease in body temperature and allergic diarrhea observed just after the OVA challenge were significantly suppressed in mice administered kaempferol.

immunology↗

bioRxiv2024

NR4A3 deficiency ameliorates contact hypersensitivity and exacerbates psoriasis by regulating gene expression in dendritic cells

NR4A3 is a transcription factor that belongs to the nuclear receptor superfamily. To reveal the roles of NR4A3 in skin diseases, we constructed contact hypersensitivity (CHS)- and imiquimod-induced psoriasis models in Nr4a3-/- mice. In the CHS induced Nr4a3-/- mice, ear swelling was significantly reduced, accompanied by suppressed migration of dendritic cells (DCs), in which CCR7 expression was reduced. In contrast, ear swelling in psoriasis model Nr4a3-/- mice was enhanced. The expression levels of inflammatory cytokines in psoriasis skin lesions and in TLR7-ligand-stimulated DCs were increased by NR4A3 deficiency. Subcutaneous (s.c.) injection of hapten-treated Nr4a3+/+ DCs induced ear swelling in Nr4a3-/- mice, and s.c. injection of TLR7-ligand-stimulated Nr4a3-/- DCs caused significant ear swelling. Compared with Nr4a3+/+ DCs, Nr4a3-/- DCs expressed lower levels of CCR7 and IRF4, and higher levels of TLR7, IRF7, IFN-{beta}, and PU.1. Taken together, NR4A3 deficiency ameliorates CHS and exacerbates psoriasis by regulating the expression of CCR7, TLR7, and related transcription factors in DCs.

immunology↗

bioRxiv2023

The gut lactic acid bacteria metabolite, 10-oxo-cis-6,trans-11-octadecadienoic acid, suppresses inflammatory bowel disease in mice by modulating the NRF2 pathway and GPCR-signaling

Various gut bacteria, including Lactobacillus plantarum, possess several enzymes that produce hydroxy fatty acids (FAs), oxo FAs, conjugated FAs, and partially saturated FAs from polyunsaturated FAs as secondary metabolites. Among these derivatives, we identified 10-oxo-cis-6,trans-11-octadecadienoic acid ({gamma}KetoC), a {gamma}-linolenic acid (GLA)-derived enon FA, as the most effective immunomodulator, which inhibited the antigen-induced immunoactivation and LPS-induced production of inflammatory cytokines. The treatment with {gamma}KetoC significantly suppressed proliferation of CD4+ T cells, LPS-induced activation of bone marrow-derived dendritic cells (BMDCs), and LPS-induced IL-6 release from peritoneal cells, splenocytes, and CD11c+ cells isolated from the spleen. {gamma}KetoC also inhibited the release of inflammatory cytokines from BMDCs stimulated with poly-I:C, R-848, or CpG. Further in vitro experiments using an agonist of GPR40/120 suggested the involvement of these GPCRs in the effects of {gamma}KetoC on DCs. We also found that {gamma}KetoC stimulated the NRF2 pathway in DCs, and the suppressive effects of {gamma}KetoC and agonist of GPR40/120 on the release of IL-6 and IL-12 were reduced in Nrf2-/- BMDCs. We evaluated the role of NRF2 in the anti-inflammatory effects of {gamma}KetoC in a dextran sodium sulfate-induced colitis model. The oral administration of {gamma}KetoC significantly reduced body weight loss, improved stool scores, and attenuated atrophy of the colon, in wild-type C57BL/6 and Nrf2+/- mice with colitis. In contrast, the pathology of colitis was deteriorated in Nrf2-/- mice even with the administration of {gamma}KetoC. Collectively, the present results demonstrated the involvement of the NRF2 pathway and GPCRs in {gamma}KetoC-mediated anti-inflammatory responses.

immunology↗

bioRxiv2023

Butyrate, valerate, and niacin ameliorate anaphylaxis by suppressing IgE-dependent mast cell activation: Roles of GPR109A, PGE2, and epigenetic regulation

Short chain fatty acids (SCFAs) were recently shown to modulate the development and functions of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) are not fully understood. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis in mice. Butyrate and valerate suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by pertussis toxin and by the knockdown of Gpr109a. A treatment with trichostatin A suppressed IgE-mediated MC activation and reduced the surface expression level of Fc{varepsilon}RI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly decreased by the treatment with PGE2 whose release from MCs was markedly enhanced by SCFAs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by COX inhibitors and the EP3 antagonist. Key MessagesShort chain fatty acids (SCFAs), particularly butyrate and valerate, suppress the IgE-mediated activation of mast cells (MCs) in vivo and in vitro. SCFAs enhance the release of PGE2 from MCs, which inhibits the IgE-mediated activation of MCs. Niacin, a ligand of GPR109A, ameliorates IgE-dependent anaphylaxis. The administration of COX inhibitors or an antagonist of PGE2 receptor 3 (EP3) inhibited the suppressive effects of butyrate and niacin on IgE-dependent anaphylaxis.

immunology↗

bioRxiv2023

Kaempferol suppresses the activation of mast cells by modulating the expression of FcϵRI and SHIP1

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of Fc{varepsilon}RI on BMMCs, but the mRNA levels of Fc{varepsilon}RI, {beta}, and {gamma}-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface Fc{varepsilon}RI on BMMCs was still observed in the presence of a protein-synthesis inhibitor. These results suggest that kaempferol reduced the surface Fc{varepsilon}RI on BMMCs, independently of transcription and translation. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NRF2, a master transcription factor of antioxidant stress, in BMMCs, inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we observed that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating Fc{varepsilon}RI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

immunology↗

bioRxiv2023

Immunostimulatory effects of Bacillus coagulans SANK70258

Specific intestinal bacteria modulate immunoresponses through various pathways, and several probiotic bacteria have been identified as immunostimulants by screening. In the present study, we evaluated the immunomodulating effects of Bacillus coagulans SANK70258 (B. coagulans SANK70258), a spore-forming and lactic acid-producing bacterium usable as food supplement for human and animals. We found that treatment of mouse splenocytes with {gamma}-ray irradiated B. coagulans SANK70258 induced high amount of IFN-{gamma} in comparison with 7 kinds of typical lactic acid bacteria. Further analyses using splenocytes revealed that NK cell is a major source of IFN-{gamma}, and B. coagulans SANK70258-induced IFN-{gamma} production was inhibited by neutralization of IL-12 or IL-23, depletion of CD11c+ cells, and inhibition of NF{kappa}B. B. coagulans SANK70258 also induced release of IFN-{gamma} from activated CD8+ T cells, and increased expression of chemokine receptors in CD8+ T cells. B. coagulans SANK70258-treatment induced production of cytokines from bone marrow-derived dendritic cells, which is reduced by knockdown of Tlr2 and Nod2. B. coagulans SANK70258-treatment also induced IgA production from Peyers patch cells with high level among tested lactic bacteria. The oral intake of {gamma}-ray irradiated B. coagulans SANK70258 significantly increased intestinal IgA levels and IgA-expressing B cells in the Peyers patch of mice. Taken together, we conclude that B. coagulans SANK70258 possesses high activity as immunostimulant inducing production of IFN-{gamma} and IgA.

immunology↗

bioRxiv2022

Morphogenesis of bullet-shaped rabies virus particles requires a functional interplay between the viral matrix protein and ESCRT-I component TSG101

Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host ESCRT (endosomal sorting complex required for transport) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection, and specifically the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a siRNA screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late-domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. ImportanceEnveloped-viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT (endosomal sorting complex required for transport) complexes. Some enveloped-viruses utilize their late (L)-domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped-virions, but the underlying molecular mechanisms involved remain elusive. Herein, we showed that TSG101, one of ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

microbiology↗

bioRxiv2022

Rose flavor compound β-damascone regulates dendritic cell-mediated immunoresponses by modulating the NRF2 pathway and ameliorates contact hypersensitivity

Dendritic cells (DCs), which are typical antigen-presenting cells, localize to various sites in the body, particularly the front line of infection as sentinels, and are involved in innate and adaptive immune responses. Although the functions of DCs, such as pathogen-induced cytokine production and antigen-specific T cell activation, are important for host defenses against infection and tumorigenesis, the hyper- and/or extended activation of DCs leads to inflammatory and autoimmune diseases. In the present study, {beta}-damascone, a major ingredient of rose fragrance, was selected from an aroma library as a candidate compound that suppresses antigen-induced immune responses. {beta}-Damascone inhibited the functions of DCs, including the antigen-dependent proliferation of T cells, DC-induced Th1 development, and the TLR ligand-induced production of inflammatory cytokines by DCs. The {beta}-damascone treatment also increased the protein level of the transcription factor NRF2, which plays key roles in antioxidant responses, and the transcription of Hmox1, a target gene of NRF2, in DCs. Nrf2-/- DCs induced Th1-development and produced large amount of IL-12p40 even in the presence of {beta}-damascone, whereas these functions by Nrf2+/- DCs were inhibited by {beta}-damascone under the same conditions. The intake of {beta}-damascone suppressed ear swelling in contact hypersensitivity (CHS) model mice, but not in CHS-induced Nrf2-/- mice. Collectively, the present results indicate the potential of the rose aroma compound {beta}-damascone, which suppresses DC-mediated immune responses by activating the NRF2 pathway in DCs, for the prevention and/or attenuation of immune-mediated diseases.

immunology↗

bioRxiv2021

Coordination of KIF3A and KIF13A regulates leading edge localization of MT1-MMP in cancer cells

MT1-MMP plays a crucial role in promoting the cellular invasion of cancer cells by degrading the extracellular matrix to create a path for migration. During this process, its localization at the leading edge of migrating cells is critical, and it is achieved by targeted transport of MT1-MMP-containing vesicles along microtubules by kinesin superfamily proteins (KIFs). Here we identified three KIFs involved in MT1-MMP vesicle transport: KIF3A, KIF13A, and KIF9. Knockdown of KIF3A and KIF13A effectively inhibited MT1-MMP-dependent collagen degradation and invasion, while knockdown of KIF9 increased collagen degradation and invasion. Our data suggest that KIF9 competes with KIF3A/KIF13A to bring MT1-MMP vesicles to different locations in the plasma membrane. Live-cell imaging analyses have indicated that KIF3A and KIF13A coordinate to transport the same MT1-MMP-containing vesicles. Taken together, we have identified a unique interplay between three KIFs to regulate leading edge localization of MT1-MMP and MT1-MMP-dependent cancer cell invasion.

cell biology↗

bioRxiv2020

Sclerostin directly stimulates osteocyte synthesis of fibroblast growth factor-23

Osteocyte produced fibroblast growth factor 23 (FGF23) is the key regulator of serum phosphate (Pi) homeostasis. The interplay between parathyroid hormone (PTH), FGF23 and other proteins that regulate FGF23 production and serum Pi levels is complex and incompletely characterised. Evidence suggests that the protein product of the SOST gene, sclerostin (SCL), also a PTH target and also produced by osteocytes, plays a role in FGF23 expression, however the mechanism for this effect is unclear. Part of the problem of understanding the interplay of these mediators is the complex multi-organ system that achieves Pi homeostasis in vivo. In the current study, we sought to address this using a unique cell line model of the osteocyte, IDG-SW3, known to express FGF23 at both the mRNA and protein levels. In cultures of differentiated IDG-SW3 cells, both PTH1-34 and recombinant human (rh) SCL remarkably induced Fgf23 mRNA expression dose-dependently within 3 hours. Both rhPTH1-34 and rhSCL also strongly induced C-terminal FGF23 protein secretion. Secreted intact FGF23 levels remained unchanged, consistent with constitutive post-translational cleavage of FGF23 in this cell model. Both rhPTH1-34 and rhSCL treatments significantly suppressed mRNA levels of Phex, Dmp1 and Enpp1 mRNA, encoding putative negative regulators of FGF23 levels, and induced Galnt3 mRNA expression, encoding N-acetylgalactosaminyl-transferase 3 (GalNAc-T3), which protects FGF23 from furin-like proprotein convertase-mediated cleavage. The effect of both rhPTH1-34 and rhSCL was antagonised by pre-treatment with the NF-{kappa}{beta} signalling inhibitors, BAY11 and TPCK. RhSCL also stimulated FGF23 mRNA expression in ex vivo cultures of human bone. These findings provide evidence for the direct regulation of FGF23 expression by sclerostin. Locally expressed sclerostin via the induction of FGF23 in osteocytes thus has the potential to contribute to the regulation of Pi homeostasis.

physiology↗