Biology subjects

Amano, M.

Publications and source records attributed to Amano, M..

10 recordsLinked to original sources

bioRxiv2026

Oribatid Mites Supply Tetrodotoxin to Poisonous Newts

Tetrodotoxin (TTX) is a potent neurotoxin widely distributed among marine and terrestrial animals, yet how terrestrial vertebrates acquire this toxin has been debated for decades. Here, we identify oribatid mites as the primary dietary sources of TTX for the Japanese fire-bellied newt, Cynops pyrrhogaster. We detect TTX and its analogs in the mites Scheloribates processus and Galumna sp. KM1, which are preferentially consumed by terrestrial newts. In a controlled toxification experiment, juvenile newts accumulated TTX after consuming TTX-bearing mites, directly demonstrating dietary sequestration. Our results demonstrate that the early terrestrial stage is critical for lifelong TTX acquisition in newts. Notably, we also detect pumiliotoxin, a poison-frog alkaloid, in S. processus, suggesting that newts and poison frogs have convergently evolved to exploit toxin-bearing microarthropods for chemical defense. We propose a "terrestrial toxic web" hypothesis in which oribatid mites act as a shared environmental reservoir linking putative microbial toxin production to vertebrate chemical defense, highlighting microarthropods as key mediators in terrestrial toxin flow.

ecology↗

bioRxiv2025

Covalent anti-HIV compound that induces HIV-1 capsid multimerization and degradation decomposes the viral core

Covalent inhibitors form strong, essentially irreversible interactions with their targets, offering enhanced structural selectivity within the target cavity and prolonged target engagement even at low doses. Here, we report that ACAi-001, an HIV-1 capsid (CA)-targeting compound identified through in silico screening of the hydrophobic cavity in the CA N-terminal domain (CA-NTD), inhibits HIV-1 replication in cell-based assays. ACAi-001 covalently binds CA to induce aberrant CA multimerization and degradation in vitro, a pharmacological property distinct from that of existing CA inhibitors such as lenacapavir. This mechanism was confirmed by Western blotting (WB), size exclusion chromatography (SEC), differential scanning fluorimetry (DSF), and liquid chromatography-mass spectrometry (LC-MS). These results suggest that ACAi-001, with two putative covalent interactions in its chemical structure, binds directly to Ser16 in the N-terminal domain targeting cavity in addition to Cys198 or Cys218 in the C-terminal domain of CA. The treatment of viral particles with ACAi-001 results in the loss of intact, conical HIV-1 cores, as directly visualized by transmission electron microscopy (TEM), establishing the HIV-1 core as the direct target of ACAi-001. These findings demonstrated that ACAi-001 may be a promising next-generation anti-HIV-1 CA inhibitor with unique mechanisms of action, offering new insight into HIV-1 core assembly and disassembly.

microbiology↗

bioRxiv2024

The crucial role of intercellular calcium wave propagation triggered by influenza A virus in promoting infection

Influenza A viruses (IAVs) initially infect a few host cells, and the infection subsequently spreads to neighboring cells. However, the molecular mechanisms underlying this expansion remain unclear. Here, we show that IAV infection upregulates the frequency of intercellular calcium wave propagations (iCWPs) that mediate the spread of IAVs. ADP released from initially infected cells mediated iCWPs via the P2Y1 receptor. The generation of iCWPs and spread of viral infection were inhibited by a P2Y1 antagonist. Enhanced endocytosis in the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection, whereas an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs. Therefore, intercellular calcium signaling is essential for early expansion and potential establishment of IAV infection, which offers a promising target for IAV prophylaxis.

cell biology↗

bioRxiv2023

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

In the mammalian brain, NMDA receptors (NMDARs) activation triggers a calcium-dependent signal transduction cascade resulting in postsynaptic remodeling and behavioral learning. However, the phosphoprotein signal flow through this transduction network is poorly understood. Here, we show that NMDAR-dependent phosphorylation drives the assembly of protein signaling complexes that regulate synaptic morphology and behavior. We performed large-scale phosphoproteomic analyses of protein kinase target proteins in successive layers of the signaling network in mouse striatal/accumbal slices. NMDARs activation resulted in the phosphorylation of 194 proteins, including Rho GTPase regulators. CaMKII-mediated phosphorylation of ARHGEF2 increased its RhoGEF activity, thereby activating the RhoA-Rho-kinase pathway. Subsequent phosphoproteomics of Rho-kinase revealed 221 protein targets, including SHANK3. Experimental validation revealed a pathway from NMDAR-dependent calcium influx through CaMKII, ARHGEF2, Rho-kinase, and SHANK3 to coordinate assembly of an actin-tethered postsynaptic complex of SHANK3/NMDAR/PSD95/DLGAP3 for spine growth and aversive learning. These findings show that NMDARs initiate metabolic phosphorylation for learning.

neuroscience↗

bioRxiv2022

Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

Intradermal delivery of self-replicating RNA (srRNA) is a promising vaccine platform. Considering that human skin temperature is around 33{degrees}C, lower than core body temperature of 37{degrees}C, we have developed an srRNA that functions optimally at skin temperature and is inactivated at or above 37{degrees}C as a safety switch. This temperature-controllable srRNA (c-srRNA), when tested as an intradermal vaccine against SARS-CoV-2, functions when injected naked without lipid nanoparticles. Unlike most currently available vaccines, c-srRNA vaccines predominantly elicit cellular immunity with little or no antibody production. Interestingly, c-srRNA-vaccinated mice produced antigen-specific antibodies upon subsequent stimulation with antigen protein. Antigen-specific antibodies were also produced when B-cell stimulation using antigen protein was followed by c-srRNA booster vaccination. Using c-srRNA, we have designed a pan-coronavirus booster vaccine that incorporates both spike receptor binding domains as viral surface proteins and evolutionarily conserved nucleoproteins as viral non-surface proteins, from both SARS-CoV-2 and MERS-CoV. It can thereby potentially immunize against SARS-CoV-2, SARS-CoV, MERS-CoV, and their variants. c-srRNA may provide a route to activate cellular immunity against a wide variety of pathogens.

immunology↗

bioRxiv2021

A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

microbiology↗

bioRxiv2021

A small molecule, ACAi-028, with anti-HIV-1 activity targets a novel hydrophobic pocket on HIV-1 capsid

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection, and an attractive therapeutic target for antivirals. We report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is one of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between a reverse transcriptase inhibitor, azidothymidine (AZT), and an integrase inhibitor, raltegravir (RAL), without any effect on RT activity, suggesting that this compound may affect HIV-1 core disassembly (uncoating). Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and non-covalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues. ImportanceThese results indicate that ACAi-028 is a novel CA inhibitor that binds to the novel hydrophobic pocket of CA-NTD. This study demonstrates that a compound targeting the new hydrophobic pocket is a promising anti-HIV-1 inhibitor. The findings presented here may offer the development of a novel class of anti-viral agents that can be used, providing HIV-1 patients with more options for Anti-retroviral therapy (ART) treatment. Despite many years of successful pharmaceutical developments in the area of anti-retroviral therapy, the prevalence of drug-resistant mutations in HIV-1, necessitates the continued development of novel agents, such as ACAi-028.

microbiology↗

bioRxiv2021

Interaction Between Phosphoinositide 3-Kinase and the VDAC2 Channel Tethers Endosomes to Mitochondria and Promotes Endosome Maturation

Intracellular organelles of mammalian cells communicate with each other during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identified voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 was found to tether endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and to promote clathrin-independent endocytosis as well as endosome maturation at membrane contact sites. With a newly developed optogenetics system to induce mitochondrion-endosome association, we found that, in addition to its structural role in such association, the pore function of VDAC2 is also required for the promotion of endosome maturation. Our findings thus uncover a previously unappreciated role of mitochondrion-endosome association in the regulation of endocytosis and endosome maturation. HighlightsO_LIThe mitochondrial protein VDAC2 binds PI3K and tethers endosomes to mitochondria C_LIO_LIVDAC2 promotes clathrin-independent endocytosis C_LIO_LIVDAC2-PI3K interaction induces acidification of endosomes associated with mitochondria C_LIO_LIThe pore function of VDAC2 also contributes to endosome maturation at contact sites C_LI

cell biology↗

bioRxiv2019

A unique conformational escape reaction of HIV-1 against an allosteric integrase inhibitor

HIV-1 integrase (IN) contributes to HIV-1 RNA binding, which is required for viral maturation. Non-catalytic site integrase inhibitors (NCINIs) have been developed as allosteric IN inhibitors, which perform anti-HIV-1 activity by disrupting IN multimerization. Here, we show that IN undergoes a novel conformational alteration to escape from NCINIs. We observed that NCINI-resistant HIV-1 variants have accumulated amino acid (AA) mutations in the IN-encoding region. We employed HPLC and thermal stability assays to show that the AA mutations affect the folding and dimerization interface of the IN catalytic core domains, resulting in severely decreased multimerization of full-length IN proteins (IN under-multimerization). The under-multimerization of IN was finally restored by HIV-1 RNA in the viral particles. Our study demonstrates that HIV-1 countervails NCINIs by IN under-multimerization as a novel escape mechanism. Our findings provide information on the understanding of IN multimerization and influence the development of unique anti-HIV-1 strategies.

microbiology↗

bioRxiv2019

Localization of BCR-ABL to stress granules contributes to its oncogenic function

The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.

cell biology↗