Biology subjects

Nakanishi, H.

Publications and source records attributed to Nakanishi, H..

12 recordsLinked to original sources

bioRxiv2026

Oxidized phosphatidylinositol impairs lysosomal membrane repair to promote ferroptosis

Ferroptosis is a regulated form of cell death driven by iron-dependent and unrestrained lipid peroxidation, which generates phospholipid hydroperoxides that cause membrane rupture. Oxidized phospholipid species, including oxidized arachidonic acid-containing phosphatidylethanolamines (PE), are abundant during ferroptosis. However, previous studies have examined only a limited number of lipid species, and it remains unclear which oxidized phospholipids consistently arise across distinct cell types and ferroptosis-inducing conditions. Here, we comprehensively profiled oxidized phospholipids generated during ferroptosis across multiple cell lines and animal models. We identified PE 18:0_20:4;O3 and phosphatidylinositol (PI) 18:0_20:4;O3 as oxidized phospholipid species that are consistently detectable across all tested ferroptosis-inducing conditions. Furthermore, oxidized phosphatidylinositol impairs the phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway, a key mechanism for lysosomal membrane repair. These results indicate that the oxidized phospholipids identified here may serve as markers of ferroptosis while also acting as bioactive mediators that compromise lysosomal membrane homeostasis and repair.

cell biology↗

bioRxiv2026

Gas-phase environment activates an alternative catabolic route in toluene-degrading Acinetobacter

Volatile aromatic compounds are important industrial feedstocks but also major environmental pollutants, highlighting the need for bioprocesses for their removal and valorization. Although gas-phase bioprocesses offer practical advantages for handling poorly water-soluble and highly volatile substrates, how gas-phase environments alter microbial metabolism remains poorly understood. Here, we investigated the effect of gas-phase conditions on toluene metabolism in the highly adhesive aromatic hydrocarbon-degrading bacterium Acinetobacter sp. Tol 5. A mutant lacking todC1, which encodes an essential component of the toluene dioxygenase, failed to grow on toluene in liquid culture but retained the ability to grow on solid media under a toluene atmosphere. Consistent with this phenotype, the mutant showed no detectable toluene degradation in the liquid phase, whereas it degraded toluene under gas-phase conditions after a prolonged lag phase. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the accumulation of o-cresol and p-cresol specifically in the mutant under toluene vapor, indicating that toluene metabolism had shifted to an alternative route involving cresol intermediates. In addition, transcriptome analysis identified strong induction of the mph operon encoding phenol monooxygenase (PMO), suggesting that PMO is a likely candidate enzyme mediating TDO-independent toluene oxidation under gas-phase conditions. Together, these results demonstrate that the gas-phase environment can activate an alternative catabolic route in Tol 5 that is not active during conventional liquid cultivation. Our findings highlight the importance of direct metabolic analysis under gas-phase conditions for understanding and designing bioprocesses using highly volatile substrates.

microbiology↗

bioRxiv2025

Molecular signature of COVID-19 prior to its exacerbation by multi-omics survey

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic due to its high transmissibility and ability to evade innate immune responses. Comprehensive characterization of the disease is essential for elucidating its pathophysiology and clinical progression. In this study, we performed multi-omics analyses of plasma samples collected from SARS-CoV-2-positive patients prior to clinical deterioration of coronavirus disease 2019 (COVID-19). These samples revealed the potential of previously reported clinical parameters, including CRP and neutrophil level, to predict COVID-19 exacerbation in the early stage. Our analysis identified a novel panel of molecules that precede the clinical manifestations associated with COVID-19 progression. These candidate biomarkers exhibited strong correlations with previously reported clinical and immunological parameters. Notably, several inflammation-related markers showed inverse associations with specific interferon subtypes, including IFN-6 and IFN-8, potentially reflecting mechanisms of SARS-CoV-2-mediated immune evasion. Our findings contribute to the understanding of virus-induced acute exacerbation and offer a valuable foundation for future pandemic research.

microbiology↗

bioRxiv2025

LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

LRRK2 is a Parkinsons disease (PD)-associated kinase that phosphorylates Rab GTPases. Hyperactivation of LRRK2, which is thought to cause PD, occurs on stressed endolysosomal membranes via CASM (conjugation of ATG8 to single membranes) and Rab, whereas the involvement of specific membrane lipids remained unclear. Here, we found that LRRK2 was potently activated upon treatment with PIKfyve inhibitors that increase cellular phosphatidylinositol 3-phosphate (PI3P) levels, and that additional treatment with the class III phosphoinositide 3-kinase (PI3K) inhibitors blocking PI3P generation suppressed LRRK2 activation. Cellular PI3P levels were indeed correlated with the LRRK2 kinase activity. CASM-induced LRRK2 activation, but not CASM itself, was suppressed by PI3K inhibitors, whereas inhibition of CASM or Rab12/Rab29 suppressed LRRK2 activation induced by PIKfyve inhibitors. LRRK2 and phospho-Rab10 were co-localized with PI3P on LAMP1-positive enlarged vacuoles, and LRRK2 directly bound PI3P in vitro via its N-terminal positively charged amino acids. Together, we propose an updated mechanism of LRRK2 activation that requires CASM, Rab and PI3P-rich membranes.

cell biology↗

bioRxiv2025

Cytosolic mannosyltransferases involved in the ER N-glycosylation pathway exhibit bacterial ancestry

N-glycosylation in eukaryotes begins with the assembly of a lipid-linked oligosaccharide on the endoplasmic reticulum membrane. As a pivotal post-translational protein modification, it is conserved across all three domains of life. However, the evolutionary origins of the N-glycosylation pathway remain a subject of ongoing debate in evolutionary biology, largely due to the limited availability of robust data regarding the evolutionary trajectories of the glycosyltransferases involved in this process. Here, we present phylogenetic analyses of the eukaryotic ALG1 and ALG2 mannosyltransferases (MTases), which are crucial for constructing the core trimannosyl Man3GlcNAc2 structure conserved in eukaryotic N-glycans. Our comprehensive phylogenetic study, combined with functional and structural analyses, suggests that the ALG2 MTase likely originated from a bacterial ancestor. This inference is further supported by the identification of sequential and functional ALG1 homologs exclusively within bacterial lineages, rather than in Asgard archaea or other archaeal groups. Our findings challenge the prevailing hypothesis that the eukaryotic N-glycosylation pathway primarily evolved from archaeal ancestors, instead suggesting a chimeric origin involving contributions from both bacterial and archaeal lineages. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/682550v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ac81a8org.highwire.dtl.DTLVardef@746054org.highwire.dtl.DTLVardef@604bdborg.highwire.dtl.DTLVardef@14235d_HPS_FORMAT_FIGEXP M_FIG C_FIG The ALG glycosyltransferase (GTases) involved in the initial stage of eukaryotic N-glycosylation on the cytosolic side of the ER membrane exhibit chimeric evolutionary origins. LUCA: Last Universal Common Ancestor; LECA: Last Eukaryotic Common Ancestor. HighlightsO_LIThe evolutionary trajectories of cytosolic ALG mannosyltransferases (MTases) were studied C_LIO_LIThe ancestor of ALG2 exhibited in the LUCA but evolved its MTase activity in bacterial lineages C_LIO_LIThe ALG1 MTase originated from bacterial lineages C_LI

molecular biology↗

bioRxiv2024

Extracellular ligand-responsive translational regulation of synthetic mRNAs using engineered receptors

mRNA drugs can encode any protein and are regarded as a promising therapeutic modality. In this study, we developed a new mRNA system allowing extracellular ligand-responsive translational regulation. This system consists of three mRNAs, two of which encode components for detecting extracellular ligands, and the other encodes the protein of interest, with a binding motif on 5UTR for translational regulation. In the presence of ligand biomolecules such as arginine vasopressin (AVP) and prostaglandin E2 (PGE2), the translation level of the protein of interest is upregulated or downregulated in a ligand biomolecule concentration-dependent manner. We showed upregulation of anti-inflammatory signaling in response to an inflammatory mediator, PGE2, demonstrating that this system allowed production of the therapeutic proteins according to the disease site environment. This system will pave the way for the next-generation mRNA drugs that self-adjust their protein production levels in response to fluctuating disease states. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/615322v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1cbe28forg.highwire.dtl.DTLVardef@874f85org.highwire.dtl.DTLVardef@175e39borg.highwire.dtl.DTLVardef@b1faca_HPS_FORMAT_FIGEXP M_FIG C_FIG mRNAs encoding regulatory components (an engineered receptor and a translational regulator) and a target mRNA are co-transfected into mammalian cells. In the absence of the ligand, the target mRNA is translationally repressed by the translational regulator. The ligand induces engineered receptor-mediated degradation of the translational regulator protein, resulting in the translational activation of the target mRNA.

synthetic biology↗

bioRxiv2024

Histone serves as an eat-me signal to induce RAGE-mediated phagocytosis

The receptor for advanced glycation end products (RAGE) is a multiligand receptor that can induce phagocytosis in both professional and nonprofessional phagocytes. We found that histones are another ligand for RAGE. Binding between histones and RAGE is increased when DNA is attached to histones. While histones are chromosomal proteins in healthy cells, they are exposed to the cell surface as a complex with DNA when cells undergo apoptosis. The phagocytosis of apoptotic cells by either professional or nonprofessional phagocytes is enhanced when histones are present on the surface of apoptotic cells. Thus, histones serve as eat-me signals. In RAGE knockout cells, the phagocytosis of apoptotic cells was not influenced by the removal of histones, indicating that RAGE is required for the removal of histones from histone-presenting cells. In RAGE knockout mice, wound healing and removal of apoptotic cells from wound sites are delayed, suggesting that RAGE-mediated phagocytosis functions under physiological conditions.

cell biology↗

bioRxiv2022

Lipid-correlated alterations in the transcriptome are enriched in several specific pathways in the postmortem prefrontal cortex of Japanese patients with schizophrenia

BackgroundSchizophrenia is a chronic relapsing psychiatric disorder that is characterized by many symptoms and has a high heritability. A previous study showed that specific lipid molecules belong to phosphatidylinositol (PI) and phosphatidylserine (PS) was reduced in the postmortem prefrontal cortex of patients with schizophrenia1. However, signaling pathways contributing to the lipid changes remain unknown. Here we performed two types of transcriptome analyses in patients with schizophrenia: an un-biased transcriptome analysis solely based on RNA-seq data and a correlation analysis between levels of gene expression and lipids. Methodology/Principal FindingsRNA-Seq analysis was performed in the postmortem prefrontal cortex from 10 subjects with schizophrenia and 5 controls. Correlation analysis between the transcriptome and lipidome from 9 subjects which are the same samples in the previous lipidomics study1 (Table 1). Extraction of differentially expressed genes (DEGs) and further sequence and functional group analysis revealed changes of gene expression levels in phosphoinositide 3-kinase (PI3K)-Akt signaling and the complement system. In addition, a correlation analysis clarified alterations in several signaling/metabolic pathways including lipid-correlated genes, most of which are not found as DEGs in transcriptome analysis alone. O_TBL View this table: org.highwire.dtl.DTLVardef@80d74eorg.highwire.dtl.DTLVardef@b780b7org.highwire.dtl.DTLVardef@1637f28org.highwire.dtl.DTLVardef@1315439org.highwire.dtl.DTLVardef@190a230_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONCharacteristics of patients from whom postmortem brain samples were obtained. Abbreviations: PMI, postmortem interval, the time that has elapsed since a person has died; DOI, duration of illness, The samples used in correlation analysis are shown by black circles. C_TABLECAPTION C_TBL ConclusionsThis study provided results of the first integrated analysis of the schizophrenia-associated transcriptome and lipidome within the PFC and revealed that lipid-correlated alterations in the transcriptome are enriched in specific pathways including PI3K-Akt signaling.

pathology↗

bioRxiv2022

Activation of Nedd4L Ubiquitin Ligase by FCHO2-generated Membrane Curvature

The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates sorting in endocytosis by mediating ubiquitination of cargo molecules, such as the epithelial sodium channel (ENaC). Defects in ENaC ubiquitination cause Liddle syndrome, a hereditary hypertension. Nedd4L is catalytically autoinhibited by an intramolecular interaction between the C2 and HECT domains, but the activation mechanism is poorly understood. Here, we show that Nedd4L is activated by membranes sculpted by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. We found that FCHO2 was required for Nedd4L-mediated ubiquitination and endocytosis of ENaC. Nedd4L co-localized with FCHO2 at clathrin-coated pits where it likely became activated. Nedd4L was specifically recruited to and activated by the FCHO2 BAR domain exogenously expressed in cells. Furthermore, we reconstituted in vitro FCHO2-induced recruitment and activation of Nedd4L. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain. Consequently, this curvature activated Nedd4L by relieving autoinhibition. Thus, we show for the first time a specific functionality (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.

cell biology↗

bioRxiv2021

Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms to control cell motility and neurite outgrowth

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent, nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knock-down phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

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

A single K+-binding site in the crystal structure of the gastric proton pump

The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 [A] of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold - the highest cation gradient known in any mammalian tissue - across the membrane.

biochemistry↗