Biology subjects

Ohba, Y.

Publications and source records attributed to Ohba, Y..

9 recordsLinked to original sources

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↗

bioRxiv2024

AFG3L2-mediated proteolysis restricts mitochondrial biogenesis and gene expression in hypoxia

Mitochondria are metabolically rewired in hypoxia when cells switch to glycolytic growth. In addition to the well-established role of transcriptional and translational programs, there is increasing evidence that post-translational mechanisms contribute to the rapid adaptation of the mitochondrial proteome to hypoxia. Here, we have used a proteomic survey to define how the m-AAA protease AFG3L2, a proteolytic complex in the inner mitochondrial membrane, regulates mitochondrial proteostasis. Our experiments identify a broad spectrum of mitochondrial substrate proteins and show that AFG3L2 is activated in hypoxia along an HIF1-mTORC1 signaling axis. AFG3L2-mediated proteolysis restricts mitochondrial biogenesis and gene expression by degrading proteins, which are involved in mitochondrial protein import, mitochondrial transcription, mRNA processing, mRNA modification and stability, and RNA granule formation. Our experiments highlight the important contribution of proteolytic rewiring of the mitochondrial proteome for the adaptation to low oxygen tension and shed new light on the pathophysiology of several neurodegenerative disorders associated with mutations in AFG3L2.

cell biology↗

bioRxiv2023

Differing structures of galactoglucomannan in eudicots and non-eudicot angiosperms

The structures of cell wall mannan hemicelluloses have changed during plant evolution. Recently, a new structure called {beta}-galactoglucomannan ({beta}-GGM) was discovered in eudicot plants. This galactoglucomannan has {beta}-(1,2)-Gal--(1,6)-Gal disaccharide branches on some mannosyl residues of the strictly alternating Glc-Man backbone. Studies in Arabidopsis revealed {beta}-GGM is related in structure, biosynthesis and function to xyloglucan. However, when and how plants acquired {beta}-GGM remains elusive. Here, we studied mannan structures in many sister groups of eudicots. All glucomannan structures were distinct from {beta}-GGM. In addition, we searched for candidate mannan {beta}-galactosyltransferases (MBGT) in non-eudicot angiosperms. Candidate AtMBGT1 orthologues from rice and Amborella did not show MBGT activity in vivo. However, the AtMBGT1 orthologue from rice showed MUR3-like xyloglucan galactosyltransferase activity in complementation analysis using Arabidopsis. Further, reverse genetic analysis revealed that the enzyme contributes to proper root growth in rice. Together, gene duplication and diversification of GT47A-VII in eudicot evolution may have been involved in the acquisition of mannan {beta}-galactosyltransferase activity. Our results indicate that {beta}-GGM is likely to be a eudicot-specific mannan.

biochemistry↗

bioRxiv2022

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

Unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants imposes us to continuous control measurement. Given the rapid spread, new Omicron subvariant named BA.5 is urgently required for characterization. Here we analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1 comprehensively. Although in vitro growth kinetics of BA.5 is comparable among the Omicron subvariants, BA.5 become much more fusogenic than BA.1 and BA.2. The airway-on-a-chip analysis showed that the ability of BA.5 to disrupt the respiratory epithelial and endothelial barriers is enhanced among Omicron subvariants. Furthermore, in our hamster model, in vivo replication of BA.5 is comparable with that of the other Omicrons and less than that of the ancestral B.1.1. Importantly, inflammatory response against BA.5 is strong compared with BA.1 and BA.2. Our data suggest that BA.5 is still low pathogenic compared to ancestral strain but evolved to induce enhanced inflammation when compared to prior Omicron subvariants.

microbiology↗

bioRxiv2021

Regulation of mitochondrial proteostasis by the proton gradient

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multi-proteomic approach to demonstrate regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+/H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.

molecular biology↗

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

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

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↗

bioRxiv2019

Folding latency of fluorescent proteins affects the mitochondrial localization of fusion protein

The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (aqGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type aqGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.

cell biology↗