Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Hirota Yuko Last modified date:2024.06.03

Assistant Professor / Division of Molecular Bioinformatics / Department of Pharmaceutical Health Care and Sciences / Faculty of Pharmaceutical Sciences

1. Yuu Miyauchi, Akane Kimura, Madoka Sawai, Keiko Fujimoto, Yuko Hirota, Yoshitaka Tanaka, Shinji Takechi, Peter I. Mackenzie, Yuji Ishii, Use of a baculovirus-mammalian cell expression-system for expression of drug-metabolizing enzymes: optimization of infection with a focus on cytochrome P450 3A4., Frontiers in Pharmacology, 10.3389/fphar.2022.832931, 13, 832931, 2022.02.
2. Hong-Bin Chen, Jorge Carlos Pineda Garcia, Shinako Arizono, Tomoki Takeda, Ren-Shi Li, Yukiko Hattori, Hiroe Sano, Yuu Miyauchi, Yuko Hirota, Yoshitaka Tanaka, Yuji Ishii, DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis., Scientific reports, 10.1038/s41598-021-97961-6, 11, 1, 18532-18532, 2021.09, Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone..
3. Yuko Hirota, Masaharu Hayashi, Yuu Miyauchi, Yuji Ishii, Yoshitaka Tanaka, Keiko Fujimoto, LAPTM4α is targeted from the Golgi to late endosomes/lysosomes in a manner dependent on the E3 ubiquitin ligase Nedd4-1 and ESCRT proteins., Biochemical and biophysical research communications, 10.1016/j.bbrc.2021.03.151, 556, 9-15, 2021.06, Lysosome-associated protein transmembrane 4α (LAPTM4α) is a four transmembrane-spanning protein primarily localized in endosomes and lysosomes and has several putative lysosomal targeting signals at its C-terminal cytoplasmic domain, including tyrosine-based motifs (YxxΦ) and PY motifs (L/PxxY). LAPTM4α has been previously shown to be ubiquitinated by the E3 ubiquitin ligase Nedd4-1 through binding to its PY motifs and sorted to lysosomes, however, the molecular mechanisms underlying the localization of LAPTM4α to endosomes/lysosomes have not yet been fully elucidated. In the present study, we show that LAPTM4α binds Nedd4-1 in a manner dependent on PY motifs, while the PY motifs and Nedd4-1 are not necessarily required for LAPTM4α ubiquitination. The binding of LAPTM4α with Nedd4-1, however, is necessary for an effective sorting of LAPTM4α from the Golgi to late endosomes/lysosomes. An unexpected finding is that LAPTM4α is localized in the lumen, but not in the limiting membrane, of late endosomes, and degraded in lysosomes over time. Interestingly, we further found that siRNA knockdown of endosomal sorting complexes required for transport (ESCRT) components that mediate sorting of ubiquitinated membrane proteins into intralumenal vesicles (ILVs) of endosomes selectively blocks the transport of LAPTM4α to endosomes. Collectively, these results suggest that trafficking of LAPTM4α from the Golgi to endosomes is promoted by the interaction with Nedd4-1, which further requires ESCRT components. Furthermore, our findings highlight a novel function for ESCRT proteins in mediating protein and/or vesicle trafficking from the Golgi to endosomes/lysosomes..
4. Yuu Miyauchi, Ken Kurohara, Akane Kimura, Madoka Esaki, Keiko Fujimoto, Yuko Hirota, Shinji Takechi, Peter I. Mackenzie, Yuji Ishii, Yoshitaka Tanaka, The carboxyl-terminal di-lysine motif is essential for catalytic activity of UDP-glucuronosyltransferase 1A9, Drug metabolism and pharmacokinetics, 10.1016/j.dmpk.2020.07.006, 35, 5, 466-474, 2020.10, UDP-Glucuronosyltransferase (UGT) is a type I membrane protein localized to the endoplasmic reticulum (ER). UGT has a di-lysine motif (KKXX/KXKXX) in its cytoplasmic domain, which is defined as an ER retention signal. However, our previous study has revealed that UGT2B7, one of the major UGT isoform in human, localizes to the ER in a manner that is independent of this motif. In this study, we focused on another UGT isoform, UGT1A9, and investigated the role of the di-lysine motif in its ER localization, glucuronidation activity, and homo-oligomer formation. Immunofluorescence microscopy indicated that the cytoplasmic domain of UGT1A9 functioned as an ER retention signal in a chimeric protein with CD4, but UGT1A9 itself could localize to the ER in a di-lysine motif-independent manner. In addition, UGT1A9 formed homo-oligomers in the absence of the motif. However, deletion of the di-lysine motif or substitution of lysines in the motif for alanines, severely impaired glucuronidation activity of UGT1A9. This is the first study that re-defines the cytoplasmic di-lysine motif of UGT as an essential peptide for retaining glucuronidation capacity..
5. Keiko Fujimoto, Sanae Nakashima, Shotaro Uchida, Riham N.S. Amen, Yuji Ishii, Yuko Hirota, Yoshitaka Tanaka, HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain, Biochemistry and Biophysics Reports, 10.1016/j.bbrep.2020.100784, 23, 2020.09, HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery..
6. Keiko Fujimoto, Shotaro Uchida, Riham N.S. Amen, Yuji Ishii, Yoshitaka Tanaka, Yuko Hirota, Lysosomal integral membrane protein LGP85 (LIMP-2) is ubiquitinated at the N-terminal cytoplasmic domain, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2020.01.095, 524, 2, 424-430, 2020.04, LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme β-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated..
7. Yuu Miyauchi, Sora Kimura, Akane Kimura, Ken Kurohara, Yuko Hirota, Keiko Fujimoto, Peter I. Mackenzie, Yoshitaka Tanaka, Yuji Ishii, Investigation of the endoplasmic reticulum localization of UDP-glucuronosyltransferase 2B7 with systematic deletion mutants, Molecular Pharmacology, 10.1124/mol.118.113902, 95, 5, 551-562, 2019.05, UDP-Glucuronosyltransferase (UGT) plays an important role in the metabolism of endogenous and exogenous compounds. UGT is a type I membrane protein, and has a dilysine motif (KKXX/KXKXX) in its C-terminal cytoplasmic domain. Although a dilysine motif is defined as an endoplasmic reticulum (ER) retrieval signal, it remains a matter of debate whether this motif functions in the ER localization of UGT. To address this issue, we generated systematic deletion mutants of UGT2B7, a major human isoform, and compared their subcellular localizations with that of an ER marker protein calnexin (CNX), using subcellular fractionation and immunofluorescent microscopy. We found that although the dilysine motif functioned as the ER retention signal in a chimera that replaced the cytoplasmic domain of CD4 with that of UGT2B7, UGT2B7 truncated mutants lacking this motif extensively colocalized with CNX, indicating dilysine motif–independent ER retention of UGT2B7. Moreover, deletion of the C-terminal transmembrane and cytoplasmic domains did not affect ER localization of UGT2B7, suggesting that the signal necessary for ER retention of UGT2B7 is present in its luminal domain. Serial deletions of the luminal domain, however, did not affect the ER retention of the mutants. Further, a cytoplasmic and transmembrane domain–deleted mutant of UGT2B7 was localized to the ER without being secreted. These results suggest that UGT2B7 could localize to the ER without any retention signal, and lead to the conclusion that the static localization of UGT results from lack of a signal for export from the ER..
8. Keiko Fujimoto, Hiroaki Ida, Yuko Hirota, Masaki Ishigai, Jun Amano, Yoshitaka Tanaka, Intracellular dynamics and fate of a humanized anti-interleukin-6 receptor monoclonal antibody, Tocilizumab, Molecular Pharmacology, 10.1124/mol.115.099184, 88, 4, 660-675, 2015.10, Tocilizumab (TCZ), a humanized anti-interleukin-6 (IL-6) receptor (IL-6R) monoclonal antibody, abrogates signal transducer protein gp130-mediated IL-6 signaling by competitively inhibiting the binding of IL-6 to the receptor, and shows clinical efficacy in autoimmune and inflammatory diseases. Despite accumulating evidence for therapeutic efficacy, the behavior and fate of TCZ at the cellular level remain largely unknown. To address this, we evaluated the endocytosis and intracellular trafficking of IL-6R in HeLa cells. The results of our study provide evidence that IL-6R is constitutively internalized from the cell surface by ligand or TCZ binding and the expression of gp130 in an independent manner and is targeted via endosomes without being significantly directed to the recycling pathway to, and degraded in, lysosomes. Furthermore, the cytoplasmic tail of IL-6R is required for constitutive endocytosis of the receptor, which is mediated by the clathrin and AP-2 complex. We further demonstrate that FcRn, whose function is to regulate the serum persistence of IgG, is confined primarily to early/recycling endosomes and rapidly transits between these compartments and late endosomes/ lysosomes without being degraded. Importantly, the expression of FcRn induces the segregation of TCZ from IL-6R, resulting in extensive colocalization of TCZ and FcRn in IL-6R-depleted endosomal compartments. Collectively, our results suggest that FcRn can accelerate the retrieval of the internalized TCZ, not only from endosomes but also from lysosomes. Our findings provide new insight into the mechanism by which the antibody internalized into cells is rescued from lysosomal degradation and into how its serum levels are maintained..
9. 廣田 有子, Shun-ichi Yamashita, Yusuke Kurihara, Xiulian Jin, Masamune Aihara, Tetsu Saigusa, KANG DONGCHON, Tomotake Kanki, Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways, Autophagy, 11, 2, 332-343, 2015.02.
10. Masamune Aihara, Xiulian Jin, Yusuke Kurihara, Yutaka Yoshida, Yuichi Matsushima, Masahide Oku, Yuko Hirota, Tetsu Saigusa, Yoshimasa Aoki, Takeshi Uchiumi, Tadashi Yamamoto, Yasuyoshi Sakai, Dongchon Kang, Tomotake Kanki, The Tor and Sin3-Rpd3 complex regulate expression of the mitophagy receptor protein Atg32, J. Cell Sci., 10.1242/​jcs.153254, 2014.05.
11. Kanae Tashiro, Mayumi Shishido, Fujimoto Keiko, Yuko Hirota, Kazuyuki Yo, Takamasa Gomi, Yoshitaka Tanaka, Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components, Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2013.11.066., 443, 1, 167-172, 2014.01.
12. Tomotake Kanki, Yusuke Kurihara, Xulian Jin, Tadahiro Goda, Yusuke Ono, Masamune Aihara, Yuko Hirota, Tetsu Sigusa, Yoshimasa Aoki, Takeshi Uchiumi, Dongchon Kang, Casein kinase 2 is essential for mitophagy., EMBO Rep., 10.1038/embor.2013.114, 14, 9, 749-845, 2013.09.
13. Kurihara, Y., Kanki, T., Aoki, Y., Hirota, Y., saigusa, T., Uchiumi, T., Kang, D., Mitophagy plays an essential role in reducing mitochondrial production of reactive oxygen species and mutation of mitochondrial DNA by maintaining mitochondrial quantity and quality in yeast., J. Biol. Chem., 287, 5, 3265-3272, 2012.01.
14. Aoki, Y., Kanki, T., Hirota, Y., Kurihara, Y., Saigusa, T., Uchiumi, T., Kang, D., Phosphorylation of Serine 114 on Atg32 mediates mitophagy, Mol. Biol. Cell, 22, 17, 3206-3217, 2011.09.
15. Amano, J., Masuyama, N., Hirota, Y., Tanaka, Y., Igawa, Y., Shiokawa, R., Okutani, T., Miyayama, T., Nanami, M., Ishigai, M., Antigen-dependent Internalization is related to Rapid Elimination from Plasma of Humanized Anti-HM1.24 Monoclonal Antibody (AHM)., Drug Metab. Dispos., 38, 12, 2339-2346, 2010.12.
16. Ohzono, C., Etoh, S., Matsumoto, M., Nakayama, KI., Hirota, Y., Tanaka, Y., Fujita, H., Nedd4-interacting protein 2, a short half-life membrane protein degraded in lysosomes, negatively controls down-regulation of connexin43., Biol. Pharm. Bull., 10.1248/bpb.33.951, 33, 6, 951-957, 2010.06.
17. Hirota, Y. and Tanaka, Y., A small GTPase, human Rab32, is required for the formation of autophagic vacuoles under basal conditions. , Cell. Mol. Life Sci., 66, 17, 2913-2932, 2009.09.
18. Ikeda, K., Hirayama, M., Hirota, Y., Asa, E., Seki, J., and Tanaka, Y., Drug-induced Phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes., Biochem. Biophys. Res. Commun., 377, 1, 268-274, 2008.12.
19. Masuyama, N., Kuronita, T., Tanaka, R., Muto, T., Hirota, Y., Takigawa, A., Fujita, H., Aso, Y., Amano, J., and Tanaka, Y. , HM1.24 is internalized from lipid rafts by clathrin-mediated endocytosis through interaction with α-adaptin., J. Biol. Chem., 10.1074, 284, 15927-15941, 2009.04.
20. Hirota, Y., Masuyama, N., Kuronita, T., Fujita, H., Himeno, M., and Tanaka, Y., Analysis of post-lysosomal compartment., Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2003.12.092, 314, 2, 306-312, 2004.02.
21. Hirosako, K., Imasato, H., Hirota, Y., Kuronita, T., Masuyama, N., Nishioka, M., Umeda, A., Fujita, H., Himeno, M., and Tanaka, Y., 3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN., Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2004.02.119, 316, 3, 845-852, 2004.04.
22. Kuronita, T., Hatano, T., Furuyama, A., Hirota, Y., Masuyama, N., Saftig, P., Himeno, M., Fujita, H., and Tanaka, Y. , The NH2-terminal transmembrane and lumenal domains of LGP85 are needed for the formation of enlarged endosomes/lysosomes., Traffic, 10.1111/j.1600-0854.2005.00325.x, 6, 10, 895-906, 2005.10.
23. Hirota, Y., Kuronita, T., Fujita, H., and Tanaka, Y., A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments., Biochem. Biophys. Res. Commun., 364, 1, 40-47, 2007.12.