Biology subjects

Sato, R.

Publications and source records attributed to Sato, R..

29 records · Page 2Linked to original sources

bioRxiv2022

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders

SLC29A3, also known as ENT3, is a lysosomal transmembrane protein that transports nucleosides from the lysosomes to the cytoplasm1. Loss-of-function mutations in SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs2,3. However, little is known about the mechanism through which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was demonstrated to depend on TLR7, which senses a combination of nucleosides and oligoribonucleotides4,5. TLR7 responded to lysosomal nucleoside storage and enhanced proliferation of Ly6Chi CX3CR1low immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Because accumulated nucleosides primarily originated from cell corpse phagocytosis, TLR7 in immature monocytes recognized nucleoside storage as lysosomal stress and increased phagocyte numbers. This non-inflammatory compensatory response is referred to as the TLR7 stress response where Syk, GSK3{beta}, {beta}-catenin, and mTORC1 serve as downstream signalling molecules. In SLC29A3 disorders, histiocytosis accompanies inflammation6,7. Nucleoside storage failed to induce pro-inflammatory cytokine production in Slc29a3-/- mice, but enhanced ssRNA-dependent pro-inflammatory cytokine production in Ly6Chi classical monocytes and peripheral macrophages, not proliferating immature monocytes. Patient-derived monocytes harbouring G208R SLC29A3 mutation showed higher survival and proliferation in the presence of M-CSF and produced larger amounts of IL-6 upon ssRNA stimulation than did those derived from healthy subjects. A TLR8 antagonist inhibited the survival/proliferation of patient-derived macrophages. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

immunology↗

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

Hydroxylation site-specific and production-dependent roles of endogenous oxysterols in cellular cholesterol homeostasis

The cholesterol metabolites oxysterols play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols has been well-established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR to those added exogenously remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR. We then show that endogenous 25-hydroxycholesterol (25-HC), 27-HC, and 24S-HC synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig proteins whereas 7a-HC has little impact on SREBP-2. The results explain the hydroxylation site-specific role of endogenous oxysterols. On the other hand, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. Moreover, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on these results, we propose that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

biochemistry↗

bioRxiv2021

Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

physiology↗

bioRxiv2020

Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7{+/-}2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

bioengineering↗

bioRxiv2019

Nucleosides drive histiocytosis in SLC29A3 disorders by activating TLR7

A lysosomal transmembrane protein SLC29A3 transports nucleosides from lysosomes to the cytoplasm. Loss-of-function mutations of the SLC29A3 gene cause lysosomal nucleoside storage in monocyte/macrophages, leading to their accumulation called histiocytosis in humans and mice. Little is known, however, about a mechanism behind nucleoside-dependent histiocytosis. TLR7, an innate immune sensors for single stranded RNA, bind and respond to nucleosides. We here show that they drive nucleoside-mediated histiocytosis. Patrolling monocyte/macrophages accumulate in the spleen of Slc29a3-/- mice but not Slc29a3-/- Tlr7-/- mice. Accumulated patrolling monocyte/macrophages stored nucleosides derived from cell corpse. TLR7 was recruited to phagosomes and activated as evidenced by TLR7-dependent phagosomal maturation. TLR7 induced hyper-responsiveness to M-CSF in Slc29a3-/- monocyte/macrophages. These results suggest that TLR7 drives histiocytosis in SLC29A3 disorders. One Sentence SummarySLC29A3 disorders are caused by activation of TLR7 with accumulated nucleosides in lysosomes.

immunology↗

bioRxiv2019

DLDG1, the Arabidopsis Homolog of the Cyanobacterial H+-Extrusion Protein, Regulates Non-Photochemical Quenching in Chloroplasts

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. Herein, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for [≥]8 h per day. DLDG1 is a homolog of the plasma-membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion, and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ.

plant biology↗

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↗

bioRxiv2018

Functional Genomics Screen with Pooled shRNA Library and Gene Expression Profiling with Extracts of Azadirachta indica Identify Potential Pathways for Therapeutic Targets in Head and Neck Squamous Cell Carcinoma

Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that the neem extracts and the associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell line treated with crude neem leaf extracts, known for their anti-tumorigenic activity. By analyzing differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing, we found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-{beta} pathway and HSF-1-related gene network. Our results indicate that neem extract simultaneously affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor.

genomics↗

bioRxiv2017

A mannitol/sorbitol receptor stimulates dietary intake in Tribolium castaneum

Perception of chemical stimuli by insects aids in accepting or rejecting food. Gustatory receptors (Grs) regulating external signals in chemosensory organs have been found in many insects. Tribolium castaneum, a major pest of stored products, possesses over 200 Gr genes. An expanded repertoire of Gr genes appears to be required for diet recognition in generalist feeders; however, it remains unclear whether T. castaneum recognizes a suite of chemicals common to many products or whether it is attracted to specific chemicals, and whether its Grs are involved in its feeding behavior. It is difficult to determine the food preference of T. castaneum based on its dietary intake due to a lack of appropriate methodology. This study established a novel dietary intake estimation method using gypsum, designated the TribUTE (Tribolium Urges To Eat) assay. T. castaneum adults were fed gypsum block without added organic compounds. Sugar preference was determined by adding sweeteners and measuring the amount of gypsum in the excreta. Mannitol was the strongest attractant of T. castaneum dietary intake; in addition, TcGr20 was responsible for mannitol and sorbitol responses in Xenopus oocyte expression, but did not respond to any other non-volatile compounds tested. The EC50 values of TcGr20 for mannitol and sorbitol were 72.6 mM and 90.6 mM, respectively, suggesting that TcGr20 is a feasible receptor for the recognition of mannitol in lower concentrations. TcGr20 was expressed in the antennae, where the perception of mannitol would occur. We examined whether TcGr20 was involved in mannitol recognition using RNAi and the TribUTE assay. The amounts of excreta in TcGr20 dsRNA-injected adults decreased significantly despite the presence of mannitol, compared to that of the control adults. Taken together, our results suggest that T. castaneum adults recognized mannitol/sorbitol using TcGr20 receptors, thereby facilitating their dietary intake.\n\nAbbreviation

biochemistry↗

bioRxiv2017

Towards Water-Free Biobanks: Long-Term Dry-Preservation At Room Temperature Of Desiccation-Sensitive Enzyme Luciferase In Air-Dried Insect Cells

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

biochemistry↗