Biology subjects

Takagi, T.

Publications and source records attributed to Takagi, T..

12 recordsLinked to original sources

bioRxiv2025

Exploration of chemical probes and conformational flexibility of GID4 - the substrate receptor of human CTLH E3 ligase complex

The application of targeted protein degradation (TPD) is currently constrained by the limited availability of low-molecular-weight molecules that can recruit E3 ligases other than CRBN (Cereblon) or VHL (Von Hippel-Lindau ligase). In this study, we present the structure-based drug design (SBDD) of high-affinity ligands that engage E3 ligase GID4 (Glucose-induced degradation protein 4) in biophysical and cellular experiments. Through structural studies and molecular modeling, we identified three clusters of compounds that induce distinct conformations of GID4. We characterized potential exit vectors and used the most promising ligand as a building block to prepare bifunctional degraders in the form of proteolysis-targeting chimeras (PROTACs). Although ternary complex formation was successful in vitro, degradation of BRD4 was not observed, highlighting the need for further optimization of the degraders. Finally, we theoretically investigated the likelihood of the identified GID4 conformations participating in protein-protein interactions mediated by molecular glue mechanisms. We believe the expanded ligand diversity discovered in this study may pave the way for tuning the selectivity and efficacy of interactions involving GID4 and its neosubstrates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/662521v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@19187e8org.highwire.dtl.DTLVardef@171abd1org.highwire.dtl.DTLVardef@1c75a5forg.highwire.dtl.DTLVardef@fe17fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Targeted degradation of GSPT1 and NEK7 by a molecular glue prodrug for treatment of HCC

Targeted Protein Degradation (TPD) technology, in the form of CRBN-modulating molecular glues, offers numerous unprecedented therapeutic benefits as evidenced by the success of approved high-value immunomodulatory imide drugs (IMiDs) such as lenalidomide and pomalidomide. Building upon these successes, we employed a small CRBN-focused library of molecular glues in a phenotypic screen against hepatocellular carcinoma (HCC) cell lines. While the original library was primarily designed to target SALL4, we identified additional CRBN substrates, including GSPT1, NEK7, and CK1, whose degradation potently induced cell death in HCC cell lines. Subsequent lead optimization efforts yielded a compound, ABS-752, which demonstrated superior in vitro and in vivo activity through the potent degradation of GSPT1. Notably, ABS-752 does not form ternary complexes with CRBN and the neosubstrates. Further investigations revealed that ABS-752 is a prodrug activated by the monoamine oxidase, VAP-1, to an aldehyde intermediate and subsequently to the active molecule, ABT-002. VAP-1, which is overexpressed in cirrhotic liver, was identified as the primary monoamine oxidase responsible for the conversion of ABS-752. ABS-752 is currently in clinical trials for the treatment of HCC.

molecular biology↗

bioRxiv2025

Repurposing of the RIPK1 selective benzooxazepin-4-one scaffold for the development of a type-III LIMK1/2 inhibitor

Benzoxazepinones have been extensively studied as exclusively selective RIP kinase 1 inhibitors. This scaffold binds as a type-III inhibitor targeting the C-out/DFG-out conformation. This inactive conformation results in a large expansion of the kinase back pocket, a conformation that has also been reported for LIM kinases. Scaffold hopping is common in the design of orthosteric kinase inhibitors, but has not been explored in the design of allosteric inhibitors, mainly due to the typically exclusive selectivity of type III inhibitors. Here, we hypothesized that the shared structural properties of LIMKs and RIPKs could lead to novel type III LIMK inhibitors using the benzoxazepinone scaffold. We report the discovery of a novel LIMK1/2 inhibitor that relies on this scaffold-based approach. The discovered compound 10 showed low nanomolar potency on LIMK1/2 and exceptional selectivity, as confirmed by a comprehensive selectivity panel with residual RIPK activity as the only off-target. The study provides one of the few examples for scaffold hopping for type-III inhibitors which are usually associated with exclusive target selectivity. Table of contents (TOC) graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/636296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@19bad48org.highwire.dtl.DTLVardef@18d8929org.highwire.dtl.DTLVardef@1280043org.highwire.dtl.DTLVardef@1296ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Osmoregulation affects elimination of microplastics in fish in freshwater and marine environments

In recent decades, microplastics (MPs) have emerged as one of the biggest environmental challenges in aquatic environments. Ingestion and toxicity of MPs in seawater (SW) and freshwater (FW) fish have been studied extensively both in field and laboratory settings. However, the basic mechanism of how fish deal with MPs in SW and FW remains unclear, although physiological conditions of fish differ significantly in the two environments. In this study, using Javanese medaka (Oryzias javanicus), a euryhaline fish which adapts readily to both SW and FW, we investigated elimination of MPs in fish in SW and FW environments. We exposed O. javanicus larvae (21 days post-hatching) to 0.25 mg/L of fluorescent polystyrene microspheres (1 {micro}m) for 24 h and then conducted an elimination test for up to 5 days. The results showed that the gut retention time (GRT) of MPs is longer in FW than in SW, indicating that MP elimination occurs more quickly in SW than in FW. However, despite shorter GRTs, higher numbers of MPs tended to be retained for a longer period in SW larvae than FW larvae. Subsequently, using a fluorescent marker, gastrointestinal fluid was found to move more rapidly in the SW group. This finding indicates that water drinking accelerates gastrointestinal fluid movement, which moves MPs through the gut in SW larvae. Beside the difference in physiological conditions, MP elimination was faster when food was available, suggesting that feeding also affects MP elimination in fish. Internal factors such as body size and intestine length were also examined, but indicated no significant difference between SW and FW. Therefore, osmoregulation and feeding both influence MP elimination in fish.

physiology↗

bioRxiv2024

A coral peptide has bactericidal activity against a global marine pathogen, Vibrio coralliilyticus

Scleractinian corals and their associated microorganisms, including endosymbiotic dinoflagellates and bacteria, constitute coral holobionts. Rising seawater temperatures weaken coral symbiotic relationships, thereby increasing susceptibility to infectious diseases and leading to disease outbreaks and subsequent population declines. The temperature-dependent coral pathogen, Vibrio coralliilyticus, poses one of the greatest threats to coral reefs because of global warming. However, coral immune defenses against this pathogen are poorly understood. We previously identified coral genes responding to V. coralliilyticus exposure by transcriptomic analysis of the reef-building coral, Acropora digitifera. Here, bioinformatic analysis identified digitiferin, a coral antimicrobial peptide (AMP), in the genome of A. digitifera. Recombinant digitiferin showed antibacterial activity against both Gram-positive and -negative bacteria. A 3D structural model and direct microscopic observations revealed that digitiferin damaged bacterial cell membranes by forming pores; however, initial growth inhibition tests in 1.5 % (w/v) NaCl showed no effect on pathogens. Because we found that digitiferin is secreted from epithelial cells into mucus and shows salt sensitivity, we hypothesized that it is active against pathogens in low-salt environments. We then investigated whether it showed antibacterial activity against pathogens in low-salt, moderate-osmolality conditions, using mannitol for osmoregulation instead of NaCl. The results showed that digitiferin exhibits bactericidal activity against V. coralliilyticus under salt-free conditions. To the best of our knowledge, this is the first study to characterize a scleractinian AMP that kills V. coralliilyticus. These findings contribute to a better understanding of coral immunity and may facilitate development of techniques to overcome coral Vibrio disease. SignificanceHealthy coral reefs are one of the most important marine ecosystems, supporting [~]25 % of all marine organisms. Coral reef ecosystems are threatened by coral infectious diseases, as rising seawater temperatures exacerbate pathogen infectivity. Thus, understanding coral immune systems is becoming increasingly important to protect coral reefs. This study reports the discovery of an antimicrobial peptide, digitiferin, from the reef-building coral, Acropora digitifera. Digitiferin is effective against Gram-positive and -negative bacteria, including the major coral pathogen, Vibrio coralliilyticus, and is secreted into mucus from ectodermal granular epithelial cells. As the first antimicrobial peptide known to have bactericidal activity against V. coralliilyticus, this discovery enhances our understanding of coral immunology and coral-pathogen interactions.

microbiology↗

bioRxiv2023

Characterization of carotenoid-producing Muricauda sp. strains isolated from the reef-building coral Galaxea fascicularis and subtropical seawater

Bacterial symbionts in corals and coral-associated zooxanthellae are attracting increasing attention as potential probiotics. Some members of the family Flavobacteriaceae are zooxanthellae-associated bacteria, which are known to protect zooxanthellae from stresses such as heat and light by producing carotenoids that reduce reactive oxygen species production. This study characterized two Flavobacteriaceae bacteria, Muricauda sp. strains ORYM1 (NBRC115792) and ORYM2 (NBRC115793), isolated from the reef-building coral Galaxea fascicularis and its surrounding seawater in Okinawa, Japan, respectively. The Muricauda sp. strain ORYM2 was isolated from subtropical seawater, and carotenoid production was assessed using ORYM2 as well as ORYM1, which was previously isolated from G. fascicularis. De novo genome sequencing revealed that both strains contain complete sets of zeaxanthin biosynthesis genes, similar to those found in other Muricauda spp. Thin-layer chromatography and high-performance liquid chromatography analyses demonstrated that ORYM1 and ORYM2 produce several carotenoids. The bacterial strains and carotenoids identified in this study provide insights into the biological roles of zooxanthellae-associated bacteria in protecting zooxanthellae and reef-building corals from environmental stresses. Statements and declarationsCompeting interests: The authors declare no competing interests.

microbiology↗

bioRxiv2023

Deciphering mechanisms of UV filter- and temperature-induced bleaching in the coral Acropora tenuis, using ecotoxicogenomics

Coral reefs are at risk of bleaching due to various environmental and anthropogenic stressors such as global warming and chemical pollutants. However, there is little understanding of stressor-specific mechanisms that cause coral bleaching. Therefore, conducting accurate ecotoxicological risk assessments and deciphering modes of action of potentially deleterious ultraviolet (UV) filters (sunscreen compounds) are crucial issues. In this study, we evaluated the toxicity and bleaching effect of benzophenone-3 (BP-3), which is widely used in sunscreen products, on the reef-building coral Acropora tenuis. Furthermore, to understand differences in UV filter- and temperature-induced bleaching, a comparative ecotoxicogenomic approach using RNA-seq was integrated into a toxicity test to clarify differences in gene expression changes induced by BP-3 and heat stress (31{degrees}C). The lethal concentration 50% (LC50) was calculated as 3.9 mg/L, indicating that emission level of BP-3 was properly controlled based on the risk assessment. Differentially expressed genes related to oxidative stress and extracellular matrix organization were involved in coral responses to both BP-3 and heat stress, but their patterns differed. Whereas immune and heat-shock responses were activated in response to heat stress, activation of a drug metabolism pathway and several signal transduction pathways were identified in BP-3 treatment groups. Our study enhances understanding of stress responses in corals induced by UV filters and thermal stress. Using potential gene markers identified in this study for eco-epidemiological surveys of stressed corals, we urgently need to develop effective countermeasures.

ecology↗

bioRxiv2022

Cleavage of the Jaw1 C-terminal region enhances its augmentative effect on the Ca2+ release via inositol 1,4,5-trisphosphate receptors

Jaw1, a tail-anchored protein with 39 carboxyl (C)-terminal amino acids, is oriented to the lumen of the endoplasmic reticulum and outer nuclear membrane. We previously reported that Jaw1, as a member of the KASH protein family, plays a role in maintaining nuclear shape via its C-terminal region. Furthermore, we recently reported that Jaw1 functions as an augmentative effector of Ca2+ release from the endoplasmic reticulum by interacting with the inositol 1,4,5-trisphosphate receptors (IP3Rs). Intriguingly, the C-terminal region is partially cleaved, meaning that Jaw1 exists in the cell in at least two forms: uncleaved and cleaved. However, the mechanism of the cleavage event and its physiological significance remain to be determined. In this study, we demonstrate that the C-terminal region of Jaw1 is cleaved after its insertion by the signal peptidase complex (SPC). Particularly, our results indicate that the SPC with the catalytic subunit SEC11A, but not SEC11C, specifically cleaves Jaw1. Furthermore, using a mutant with a deficit in the cleavage event, we demonstrate that the cleavage event enhances the augmentative effect of Jaw1 on the Ca2+ release ability of IP3Rs. Summary statementThe C-terminal region of Jaw1, a tail-anchored protein, is cleaved by signal peptidase complex and this cleavage event enhances the augmentative effect of Jaw1 on the Ca2+ release activity of inositol 1,4,5-trisphosphate receptors

cell biology↗

bioRxiv2021

Mutational effects of ubiquitously present gamma radiation on Arabidopsis thaliana: insight into radiosensitivity in the reproductive stage

Earth has always been exposed to ionizing radiation from natural sources, and man-made sources have added to this radiation. In order to assess mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life-cycle of Arabidopsis thaliana under controlled conditions. Resequencing data from 12 M1 lines and 36 M2 progeny derived under gamma-irradiation conditions ranging from 0.0 to 2.0 Gy/d were obtained to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and a low-to-middling dose of radiation was assessed by statistical modeling. The increasing of de novo mutations in response to doses of irradiation fit the negative binomial model, accounting for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, with deletions being more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation are more intensive during the reproductive stage. These outcomes could provide valuable insights into practical strategies for environmental radioprotection of plants on Earth and in space.

genetics↗

bioRxiv2020

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

microbiology↗

bioRxiv2019

Nuclear actin cable with multiple branches during yeast meiosis

Actin polymerizes to form filaments/cables for motility, transport, and structural framework in a cell. Recent studies show that actin polymers are present not only in cytoplasm, but also in nuclei of vertebrate cells, and their formation is induced in response to stress. Here, by electron microscopic observation with rapid freezing and high-pressure freezing, we found a unique bundled structure containing actin in nuclei of budding yeast cells undergoing meiosis. The nuclear bundle/cable during meiosis consists of multiple filaments with a rectangular lattice arrangement often showing "feather-like" appearance. The bundle is immuno-labeled with anti-actin antibody and sensitive to an actin-depolymerizing drug. Like cytoplasmic bundles, nuclear bundles with actin are rarely seen in pre-meiotic cells and spores, and are induced during meiotic prophase-I. The formation of the nuclear bundles/cables is independent of meiotic DNA double-stranded breaks. We speculate that nuclear bundles/cables containing actin play a role in nuclear events during meiotic prophase I.

cell biology↗

bioRxiv2016

Lower brain pH as a shared endophenotype of psychotic disorders

Lower pH is a well-replicated finding in the postmortem brains of patients with schizophrenia and bipolar disorder. Interpretation of the data, however, is controversial as to whether this finding reflects a primary feature of the diseases or is a result of confounding factors such as medication, postmortem interval, and agonal state. To date, systematic investigation of brain pH has not been undertaken using animal models, which can be studied without confounds inherent in human studies. In the present study, we first confirmed that the brains of patients with schizophrenia and bipolar disorder exhibit lower pH values by conducting a meta-analysis of existing datasets. We then utilized neurodevelopmental mouse models of psychiatric disorders in order to test the hypothesis that lower brain pH exists in these brains compared to controls due to the underlying pathophysiology of the disorders. We measured pH, lactate levels, and related metabolite levels in brain homogenates from three mouse models of schizophrenia (Schnurri-2 KO, forebrain-specific calcineurin KO, and neurogranin KO mice) and one of bipolar disorder (Camk2a HKO mice), and one of autism spectrum disorders (Chd8 HKO mice). All mice were drug-naive with the same postmortem interval and agonal state at death. Upon postmortem examination, we observed significantly lower pH and higher lactate levels in the brains of model mice relative to controls. There was a significant negative correlation between pH and lactate levels. These results suggest that lower pH associated with increased lactate levels is a pathophysiology of such diseases rather than mere artifacts.

neuroscience↗