Papers - Ishida, Hanako

Cryo-EM structures of the zinc transporters ZnT3 and ZnT4 provide insights into their transport mechanisms.

Ishida H, Yo R, Zhang Z, Shimizu T, Ohto U

FEBS letters  2024.10

Research paper (scientific journal), Lead author, Accepted,  ISSN  0014-5793

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Zinc transporters (ZnTs) act as H+/Zn2+ antiporters, crucial for zinc homeostasis. Brain-specific ZnT3 expressed in synaptic vesicles transports Zn2+ from the cytosol into vesicles and is essential for neurotransmission, with ZnT3 dysfunction associated with neurological disorders. Ubiquitously expressed ZnT4 localized to lysosomes facilitates the Zn2+ efflux from the cytosol to lysosomes, mitigating the cell injury risk. Despite their importance, the structures and Zn2+ transport mechanisms remain unclear. We characterized the three-dimensional structures of human ZnT3 (inward-facing) and ZnT4 (outward-facing) using cryo-electron microscopy. By combining these structures, we assessed the conformational changes that could occur within the transmembrane domain during Zn2+ transport. Our results provide a structural basis for a more comprehensive understanding of the H+/Zn2+ exchange mechanisms exhibited by ZnTs.

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Structure of the bile acid transporter and HBV receptor NTCP

Asami J., Kimura K.T., Fujita-Fujiharu Y., Ishida H., Zhang Z., Nomura Y., Liu K., Uemura T., Sato Y., Ono M., Yamamoto M., Noda T., Shigematsu H., Drew D., Iwata S., Shimizu T., Nomura N., Ohto U.

Nature (Nature)  606 ( 7916 ) 1021 - 1026 2022.06

Research paper (scientific journal), Accepted,  ISSN  00280836

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Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus–transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.

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Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation

Ohto U., Kamitsukasa Y., Ishida H., Zhangami K., Hirama C., Maekawa S., Shimizu T. Z., Murak

Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America)  119 ( 11 ) e2121353119 2022.03

Research paper (scientific journal), Accepted,  ISSN  00278424

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The nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) responds to a vast variety of stimuli, and activated NLRP3 forms an inflammasome, which in turn is associated with conditions such as atherosclerosis, Alzheimer's disease, and diabetes. A multilayered regulatory mechanism ensures proper NLRP3 inflammasome activation, although the structural basis for this process remains unclear. This study aimed to investigate the cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950. The inhibitor binds to the cavity distinct from the nucleotide binding site in the NACHT domain and stabilizes the closed conformation of NLRP3. The barrel-shaped dodecamer composed of the inactive form of NLRP3 is formed mainly through LRR-LRR interactions on the lateral side, and the highly positively charged top and bottom sides composed of NACHT domains provide a scaffold for membrane association. The cryo-electron microscopy structure suggests that oligomerization of NLRP3 is necessary for its membrane association; it is subsequently disrupted for activation, hence serving as a key player in controlling the spatiotemporal NLRP3 inflammasome activation. These findings are expected to contribute to the development of drugs targeting NLRP3 in future.

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Cryo-EM structures of Toll-like receptors in complex with UNC93B1

Ishida H., Asami J., Zhang Z., Nishizawa T., Shigematsu H., Ohto U., Shimizu T.

Nature Structural and Molecular Biology (Nature Structural and Molecular Biology)  28 ( 2 ) 173 - 180 2021.02

Research paper (scientific journal), Lead author, Accepted,  ISSN  15459993

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Nucleic acid–sensing Toll-like receptors (TLRs) play a pivotal role in innate immunity by recognizing foreign DNA and RNA. Compartmentalization of these TLRs in the endosome limits their activation by self-derived nucleic acids and reduces the possibility of autoimmune reactions. Although chaperone Unc-93 homolog B1, TLR signaling regulator (UNC93B1) is indispensable for the trafficking of TLRs from the endoplasmic reticulum to the endosome, mechanisms of UNC93B1-mediated TLR regulation remain largely unknown. Here, we report two cryo-EM structures of human and mouse TLR3–UNC93B1 complexes and a human TLR7–UNC93B1 complex. UNC93B1 exhibits structural similarity to the major facilitator superfamily transporters. Both TLRs interact with the UNC93B1 amino-terminal six-helix bundle through their transmembrane and luminal juxtamembrane regions, but the complexes of TLR3 and TLR7 with UNC93B1 differ in their oligomerization state. The structural information provided here should aid in designing compounds to combat autoimmune diseases.

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The effect of anesthetics on toll like receptor 9.

Koutsogiannaki S, Bu W, Hou L, Shibamura-Fujiogi M, Ishida H, Ohto U, Eckenhoff RG, Yuki K

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 34 ( 11 ) 14645 - 14654 2020.11

Research paper (scientific journal), Accepted,  ISSN  0892-6638

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Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Kanoh H, Nitta T, Go S, Inamori KI, Veillon L, Nihei W, Fujii M, Kabayama K, Shimoyama A, Fukase K, Ohto U, Shimizu T, Watanabe T, Shindo H, Aoki S, Sato K, Nagasaki M, Yatomi Y, Komura N, Ando H, Ishida H, Kiso M, Natori Y, Yoshimura Y, Zonca A, Cattaneo A, Letizia M, Ciampa M, Mauri L, Prinetti A, Sonnino S, Suzuki A, Inokuchi JI

The EMBO journal 39 ( 12 ) e101732 2020.06

Research paper (scientific journal), Accepted,  ISSN  0261-4189

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Structural analysis of HTL and D14 proteins reveals the basis for ligand selectivity in Striga.

Xu Y, Miyakawa T, Nosaki S, Nakamura A, Lyu Y, Nakamura H, Ohto U, Ishida H, Shimizu T, Asami T, Tanokura M

Nature communications 9 ( 1 ) 3947 2018.09

Research paper (scientific journal), Accepted

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Structural basis for species-specific activation of mouse Toll-like receptor 9.

Ishida H, Ohto U, Shibata T, Miyake K, Shimizu T

FEBS letters 592 ( 15 ) 2636 - 2646 2018.08

Research paper (scientific journal), Lead author, Accepted,  ISSN  0014-5793

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Toll-like Receptor 9 Contains Two DNA Binding Sites that Function Cooperatively to Promote Receptor Dimerization and Activation.

Ohto U, Ishida H, Shibata T, Sato R, Miyake K, Shimizu T

Immunity 48 ( 4 ) 649 - 658.e4 2018.04

Research paper (scientific journal), Accepted,  ISSN  1074-7613

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Structure of IZUMO1-JUNO reveals sperm-oocyte recognition during mammalian fertilization.

Ohto U, Ishida H, Krayukhina E, Uchiyama S, Inoue N, Shimizu T

Nature 534 ( 7608 ) 566 - 9 2016.06

Research paper (scientific journal), Accepted,  ISSN  0028-0836

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Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9.

Ohto U, Shibata T, Tanji H, Ishida H, Krayukhina E, Uchiyama S, Miyake K, Shimizu T

Nature 520 ( 7549 ) 702 - 5 2015.04

Research paper (scientific journal), Accepted,  ISSN  0028-0836

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