Biology subjects

Osada, H.

Publications and source records attributed to Osada, H..

9 recordsLinked to original sources

bioRxiv2026

PLCXD2 preferentially hydrolyzes phosphatidylinositol and supports retinal lipid homeostasis and photoreceptor integrity

PLCXD proteins have been linked to phosphoinositide metabolism for more than a decade, and PLCXD2 activity has been associated with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] depletion, yet its preferred direct substrate has remained unresolved. Here, cellular lipidomics showed that catalytically active PLCXD2 reduced the major phosphatidylinositol (PI) 38:4 species, with more modest phosphoinositide changes and increased diacylglycerol (DAG) and phosphatidic acid (PA). Using recombinant PLCXD2 and acyl-chain-matched substrates, we found that PLCXD2 hydrolyzed PI substantially more efficiently than PI(4,5)P2 and most other phosphoinositides tested. In Plcxd2-deficient retina, PI abundance was preserved, whereas DAG and PA were reduced without broad changes in major membrane phospholipid classes. PLCXD2 loss was associated with early structural abnormalities at the photoreceptor-bipolar synaptic interface, followed by outer nuclear layer thinning and reduced electroretinographic a-wave responses. These findings identify PLCXD2 as a PI-preferring mammalian PLC and reveal an endogenous role in retinal lipid homeostasis and outer retinal integrity.

biochemistry↗

bioRxiv2025

Population-scale chemical response revealed by a barcoded yeast collection

Natural genetic variation shapes how microbial populations adapt to environmental and chemical challenges, but scalable approaches to map genotype-phenotype relationships across diverse genetic backgrounds remain limited. Here, we developed a systematically barcoded collection of 520 Saccharomyces cerevisiae natural isolates that captures the ecological, geographical and genetic diversity of the species. Using pooled barcode sequencing, we profiled fitness responses to over 600 bioactive and natural compounds, revealing broader and more polarized bioactivity than the standard yeast gene-deletion collection. Fitness-based clustering defined six major compound groups with reproducible, population-structured sensitivity patterns. Genome-wide association analysis identified significant genetic variants across 107 compounds, linking natural polymorphisms to chemical responses and involving genes in genome maintenance, ribosome biogenesis, vesicular trafficking and stress tolerance. Together, our barcoded natural population provides a scalable framework for chemical-genetic screening, enabling systematic dissection of how genetic diversity shapes microbial fitness and adaptation.

evolutionary biology↗

bioRxiv2023

Quinoclamine inhibits Shiga toxin production in enterohemorrhagic Escherichia coli

ObjectivesEnterohemorrhagic Escherichia coli (EHEC) is responsible for the most severe symptoms of E. coli infections, including hemorrhagic colitis and hemorrhagic uremic syndrome. Shiga toxin 2 (Stx2) plays a significant role as a major virulence factor. The genes encoding Stx2 locate in lambda-like prophage on the EHEC genome. Consequently, Stx2 is expressed when production of the phage is induced by the SOS response. Antibiotic treatment is not recommended for curing the bacterial infection, because it is associated with severe hemorrhagic uremic syndrome. If Stx2 production is prevented, EHEC pathogenicity significantly decreases, and antibiotics may be available to treat the infection. MethodsWe conducted two independent screenings to identify Stx2 production inhibitors for libraries from the RIKEN Natural Product Depository (NPDepo); namely, screening of the Authentic Library, and two-round screening of the Pilot and Analog Libraries. ResultsThe screening of Authentic Library identified niclosamide as a Stx2 production inhibitor. Besides, two naphthoquinoids were identified after the two-round of screening of the Pilot and Analog Libraries. Niclosamide, and quinoclamine, which has structure shared in the two naphthoquinoids, prevented cell lysis via the phage production and ceased Stx2 production in EHEC. The SOS reporter assay indicated that quinoclamine prevented the SOS response in E. coli, whereas niclosamide did not. ConclusionsThese findings suggest that quinoclamine inhibited Stx2 production by preventing the SOS response, whereas niclosamide was involved in phage propagation following the SOS response. These compounds can be a potential therapeutic option to treat EHEC infections.

microbiology↗

bioRxiv2022

Identification of a novel family of benzimidazole species-selective Complex I inhibitors as potential anthelmintics

Soil-transmitted helminths (STHs) including Ascaris, hookworm, and whipworm are major human pathogens infecting over a billion people worldwide1,2. There are few existing classes of anthelmintics and resistance is increasing3-5 -- there is thus an urgent need for new classes of these drugs. Here we focus on identifying compounds that interfere with the unusual anaerobic metabolism that STHs use to survive the highly hypoxic conditions of the host gut6-9. This requires rhodoquinone (RQ), a quinone electron carrier that is not made or used by the STH hosts10. We previously showed that C. elegans also uses this rhodoquinone-dependent metabolism (RQDM)11 and established a high throughput assay for RQDM11. We screened a collection of 480 natural products for compounds that kill worms specifically when they rely on RQDM -- these 480 are representatives of a full library of ~25,000 natural products and derivatives12,13. We identify several classes of compound including a novel family of species selective inhibitors of Complex I. These Complex I inhibitors are based on a benzimidazole core but unlike commercial benzimidazole anthelmintics they do not target microtubules14-17. We screened over 1,200 benzimidazoles and identify the key structural requirements for species selective Complex I inhibition. We suggest that these novel benzimidazole species-selective Complex I inhibitors may be potential anthelmintics.

molecular biology↗

bioRxiv2021

Violet light modulates the central nervous system to regulate memory and mood

Light stimuli from the external environment serves as a signal. Photoreceptors receive photons at the outer nuclear layer of the retina. Non-visual photoreceptors, such as opsin5 (also known as OPN5 or neuropsin), are expressed in the retinal ganglion cells (RGCs) and hypothalamus to regulate the circadian cycle and body temperature. Here, we show that violet light (VL) stimuli received by OPN5-positive RGCs are transmitted to the habenula brain region. VL improves memory in aged mice and simultaneously increases neural architecture-related genes such as oligodendrocyte-related genes in the hippocampus. In addition, VL improves depressive-like behaviors in the social defeat stress model in an OPN5 dependent manner. Following VL exposure, cFos activation is observed at the nucleus accumbens (NAc) and the paraventricular thalamic nucleus (PVT). Taken together, the results indicate that violet light modulates brain function such as memory and mood by transmitting the signal from RGCs to the habenula region in the brain.

neuroscience↗

bioRxiv2021

Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Molecular chirality, discovered by Louis Pasteur in the middle of the 19th century1, is found in most primary and secondary metabolites. Particularly, the so-called natural products are rich in chiral centres2. The stereochemistry of natural products is strictly recognized in living organisms, and is thus closely related to their biological functions. The Diels-Alder (DA) reaction, which forms a six-membered ring with up to four chiral centres, is a fundamental practical reaction for C-C bond formation in synthetic chemistry3. Nature has also adopted this reaction to elaborate the complex structures of natural products using enzymes derived from various progenitor proteins4-7. Although enzymes catalysing the DA reaction, Diels-Alderases (DAases), have attracted increasing attention, little is known about the molecular mechanism by which they control the stereochemistry and perform catalysis. Here, we solved the X-ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyse intramolecular DA reactions to form enantiomeric decalin scaffolds during biosynthesis of the HIV-1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin8,9. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided dynamic binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, explaining the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyse the stereoselective DA reaction. This study highlights the distinct molecular mechanisms of the enzymatic DA reaction and its stereoselectivity experimentally and computationally. We anticipate that clarified molecular mechanism herein provides not only the basic understanding how these important enzymes work but also the guiding principle to create artificial enzymes that produce designer bioactive molecules.

biochemistry↗

bioRxiv2020

Oxicam-type nonsteroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway

Salicylic acid (SA) and its structural analogs are nonsteroidal anti-inflammatory drugs (NSAIDs) that target mammalian cyclooxygenases. In plants, SA acts as a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identified a number of NSAIDs that enhance bacterial effector-induced cell death. Among them, the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent immune responses in plants. TNX treatment reduces NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. Surprisingly, however, cysteine modification associated with NPR1 oligomerization via intermolecular disulfide bonds is not affected by either SA or TNX. Therefore, oxicam-type NSAIDs highlight importance of SA effects on the cytosolic redox status, but not on cysteine modification or oligomerization of NPR1.

plant biology↗

bioRxiv2017

Large-scale interpretation of chemical-genetic interaction profiles using a genetic interaction network

Chemical-genetic interactions - observed when the treatment of mutant cells with chemical compounds reveals unexpected phenotypes - contain rich functional information linking compounds to their cellular modes of action. To systematically identify these interactions, an array of mutants is challenged with a compound and monitored for fitness defects, generating a chemical-genetic interaction profile that provides a quantitative, unbiased description of the cellular function(s) perturbed by the compound. Genetic interactions, obtained from genome-wide double-mutant screens, provide a key for interpreting the functional information contained in chemical-genetic interaction profiles. Despite the utility of this approach, integrative analyses of genetic and chemical-genetic interaction networks have not been systematically evaluated. We developed a method, called CG-TARGET (Chemical Genetic Translation via A Reference Genetic nETwork), that integrates large-scale chemical-genetic interaction screening data with a genetic interaction network to predict the biological processes perturbed by compounds. CG-TARGET compared favorably to a baseline enrichment approach across a variety of benchmarks, achieving similar accuracy while substantially improving the ability to control the false discovery rate of biological process predictions. We applied CG-TARGET to a recent screen of nearly 14,000 chemical compounds in Saccharomyces cerevisiae, integrating this dataset with the global S. cerevisiae genetic interaction network to prioritize over 1500 compounds with high-confidence biological process predictions for further study. Upon investigation of the compatibility of chemical-genetic and genetic interaction profiles, we observed that one-third of observed chemical-genetic interactions contributed to the highest-confidence biological process predictions and that negative chemical-genetic interactions overwhelmingly formed the basis of these predictions. We present here a detailed characterization of the CG-TARGET method along with experimental validation of predicted biological process targets, focusing on inhibitors of tubulin polymerization and cell cycle progression. Our approach successfully demonstrates the use of genetic interaction networks in the functional annotation of compounds to biological processes.

systems biology↗

bioRxiv2017

Functional Annotation of Chemical Libraries across Diverse Biological Processes

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells to a compound, revealing chemical-genetic interactions that can elucidate a compounds mode of action. We developed a highly parallel and unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized, diagnostic mutant collection that is predictive all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode sequencing protocol, enabling assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened 7 different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

systems biology↗