研究者詳細 - 上原　孝

論文 - 上原　孝

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Pivotal role for S-nitrosylation of DNA methyltransferase 3B in epigenetic regulation of tumorigenesis. 国際誌

Kosaku Okuda, Kengo Nakahara, Akihiro Ito, Yuta Iijima, Ryosuke Nomura, Ashutosh Kumar, Kana Fujikawa, Kazuya Adachi, Yuki Shimada, Satoshi Fujio, Reina Yamamoto, Nobumasa Takasugi, Kunishige Onuma, Mitsuhiko Osaki, Futoshi Okada, Taichi Ukegawa, Yasuo Takeuchi, Norihisa Yasui, Atsuko Yamashita, Hiroyuki Marusawa, Yosuke Matsushita, Toyomasa Katagiri, Takahiro Shibata, Koji Uchida, Sheng-Yong Niu, Nhi B Lang, Tomohiro Nakamura, Kam Y J Zhang, Stuart A Lipton, Takashi Uehara

Nature communications 14 ( 1 ) 621 - 621 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-L-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 ≤ 100 nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B.

DOI： 10.1038/s41467-023-36232-6

Discovery of a Compound That Inhibits IRE1α S-Nitrosylation and Preserves the Endoplasmic Reticulum Stress Response under Nitrosative Stress. 国際誌

Haruna Kurogi, Nobumasa Takasugi, Sho Kubota, Ashutosh Kumar, Takehiro Suzuki, Naoshi Dohmae, Daisuke Sawada, Kam Y J Zhang, Takashi Uehara

ACS chemical biology 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes S-nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, S-nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the S-nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the S-nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified S-nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy.

DOI： 10.1021/acschembio.4c00403

Epigenetic Regulation of CXC Chemokine Expression by Environmental Electrophiles Through DNA Methyltransferase Inhibition. 国際誌

Tomoki Tsuchida, Sho Kubota, Shizuki Kamiuezono, Nobumasa Takasugi, Akihiro Ito, Yoshito Kumagai, Takashi Uehara

International journal of molecular sciences 25 ( 21 ) 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Ubiquitously distributed environmental electrophiles covalently modify DNA and proteins, potentially leading to adverse health effects. However, the impacts of specific electrophiles on target proteins and their physiological roles remain largely unknown. In the present study, we focused on DNA methylation, which regulates gene expression and physiological responses. A total of 45 environmental electrophiles were screened for inhibitory effects on the activity of DNA methyltransferase 3B (DNMT3B), a key enzyme in DNA methylation, and four compounds were identified. We focused on 1,2-naphthoquinone (1,2-NQ), an air pollutant whose toxicity has been reported previously. Interestingly, we found that 1,2-NQ modified multiple lysine and histidine residues in DNMT3B, one of which was near the active site in DNMT3B. It was found that 1,2-NQ altered gene expression and evoked inflammatory responses in lung adenocarcinoma cell lines. Furthermore, we found that 1,2-NQ upregulated CXCL8 expression through DNA demethylation of the distal enhancer and promoted cancer cell growth. Our study reveals novel mechanisms of epigenetic regulation by environmental electrophiles through the inhibition of DNMT3B activity and suggests their physiological impact.

DOI： 10.3390/ijms252111592

Therapeutic potential of 4-phenylbutyric acid against methylmercury-induced neuronal cell death in mice 国際誌

Ryohei Miki, Ryosuke Nomura, Yuta Iijima, Sho Kubota, Nobumasa Takasugi, Takao Iwawaki, Masatake Fujimura, Takashi Uehara

Archives of Toxicology 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

Abstract

Methylmercury (MeHg) is an environmental neurotoxin that induces damage to the central nervous system and is the causative agent in Minamata disease. The mechanisms underlying MeHg neurotoxicity remain largely unknown, and there is a need for effective therapeutic agents, such as those that target MeHg-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which is activated as a defense mechanism. We investigated whether intraperitoneal administration of the chemical chaperone, 4-phenylbutyric acid (4-PBA), at 120 mg/kg/day can alleviate neurotoxicity in the brains of mice administered 50 ppm MeHg in drinking water for 5 weeks. 4-PBA significantly reduced MeHg-induced ER stress, neuronal apoptosis, and neurological symptoms. Furthermore, 4-PBA was effective even when administered 2 weeks after the initiation of exposure to 30 ppm MeHg in drinking water. Our results strongly indicate that ER stress and the UPR are key processes involved in MeHg toxicity, and that 4-PBA is a novel therapeutic candidate for MeHg-induced neurotoxicity.

DOI： 10.1007/s00204-024-03902-3

その他リンク： https://link.springer.com/article/10.1007/s00204-024-03902-3/fulltext.html

Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells

Yuto Moriya, Sho Kubota, Yuta Iijima, Nobumasa Takasugi, Takashi Uehara

Biological and Pharmaceutical Bulletin 47 ( 6 ) 1119 - 1122 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pharmaceutical Society of Japan

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

DOI： 10.1248/bpb.b24-00241

Characterization of pathological changes in the olfactory system of mice exposed to methylmercury 国際誌

Yuta Iijima, Ryohei Miki, Nobumasa Takasugi, Masatake Fujimura, Takashi Uehara

Archives of Toxicology 98 ( 4 ) 1163 - 1175 2024年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

Abstract

Methylmercury (MeHg) is a well-known environmental neurotoxicant that causes severe brain disorders such as Minamata disease. Although some patients with Minamata disease develop olfactory dysfunction, the underlying pathomechanism is largely unknown. We examined the effects of MeHg on the olfactory system using a model of MeHg poisoning in which mice were administered 30 ppm MeHg in drinking water for 8 weeks. Mice exposed to MeHg displayed significant mercury accumulation in the olfactory pathway, including the nasal mucosa, olfactory bulb, and olfactory cortex. The olfactory epithelium was partially atrophied, and olfactory sensory neurons were diminished. The olfactory bulb exhibited an increase in apoptotic cells, hypertrophic astrocytes, and amoeboid microglia, mainly in the granular cell layer. Neuronal cell death was observed in the olfactory cortex, particularly in the ventral tenia tecta. Neuronal cell death was also remarkable in higher-order areas such as the orbitofrontal cortex. Correlation analysis showed that neuronal loss in the olfactory cortex was strongly correlated with the plasma mercury concentration. Our results indicate that MeHg is an olfactory toxicant that damages the central regions involved in odor perception. The model described herein is useful for analyzing the mechanisms and treatments of olfactory dysfunction in MeHg-intoxicated patients.

DOI： 10.1007/s00204-024-03682-w

その他リンク： https://link.springer.com/article/10.1007/s00204-024-03682-w/fulltext.html

Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling 査読国際誌

Atsushi Morimoto, Nobumasa Takasugi, Yuexuan Pan, Sho Kubota, Naoshi Dohmae, Yumi Abiko, Koji Uchida, Yoshito Kumagai, Takashi Uehara

Journal of Biological Chemistry 300 ( 3 ) 105679 - 105679 2024年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as Cys in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed PI3K-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K-Akt signaling by MVK reversed epidermal growth factor-induced negative regulation of autophagy and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

DOI： 10.1016/j.jbc.2024.105679

Nuclear SphK2/S1P signaling is a key regulator of ApoE production and Aβ uptake in astrocytes 国際誌

Masato Komai, Yuka Noda, Atsuya Ikeda, Nanaka Kaneshiro, Yuji Kamikubo, Takashi Sakurai, Takashi Uehara, Nobumasa Takasugi

Journal of Lipid Research 65 ( 3 ) 100510 - 100510 2024年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier BV

The link between changes in astrocyte function and the pathological progression of Alzheimer's disease (AD) has attracted considerable attention. Interestingly, activated astrocytes in AD show abnormalities in their lipid content and metabolism. In particular, the expression of apolipoprotein E (ApoE), a lipid transporter, is decreased. Because ApoE has anti-inflammatory and amyloid β (Aβ)-metabolizing effects, the nuclear receptors, retinoid X receptor (RXR) and LXR, which are involved in ApoE expression, are considered promising therapeutic targets for AD. However, the therapeutic effects of agents targeting these receptors are limited or vary considerably among groups, indicating the involvement of an unknown pathological factor that modifies astrocyte and ApoE function. Here, we focused on the signaling lipid, sphingosine-1-phosphate (S1P), which is mainly produced by sphingosine kinase 2 (SphK2) in the brain. Using astrocyte models, we found that upregulation of SphK2/S1P signaling suppressed ApoE induction by both RXR and LXR agonists. We also found that SphK2 activation reduced RXR binding to the APOE promoter region in the nucleus, suggesting the nuclear function of SphK2/S1P. Intriguingly, suppression of SphK2 activity by RNA knockdown or specific inhibitors upregulated lipidated ApoE induction. Furthermore, the induced ApoE facilitates Aβ uptake in astrocytes. Together with our previous findings that SphK2 activity is upregulated in AD brain and promotes Aβ production in neurons, these results indicate that SphK2/S1P signaling is a promising multifunctional therapeutic target for AD that can modulate astrocyte function by stabilizing the effects of RXR and LXR agonists, and simultaneously regulate neuronal pathogenesis.

DOI： 10.1016/j.jlr.2024.100510

Methylmercury-induced brain neuronal death in CHOP-knockout mice

Yuta Iijima, Ryohei Miki, Masatake Fujimura, Seiichi Oyadomari, Takashi Uehara

The Journal of Toxicological Sciences 49 ( 2 ) 55 - 60 2024年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Toxicology

Apoptosis is one of the hallmarks of MeHg-induced neuronal cell death; however, its molecular mechanism remains unclear. We previously reported that MeHg exposure induces neuron-specific ER stress in the mouse brain. Excessive ER stress contributes to apoptosis, and CHOP induction is considered to be one of the major mechanisms. CHOP is also increased by MeHg exposure in the mouse brain, suggesting that it correlates with increased apoptosis. In this study, to clarify whether CHOP mediates MeHg-induced apoptosis, we examined the effect of CHOP deletion on MeHg exposure in CHOP-knockout mice. Our data showed that CHOP deletion had no effect on MeHg exposure-induced weight loss or hindlimb impairment in mice, nor did it increase apoptosis or inhibit neuronal cell loss. Hence, CHOP plays little role in MeHg toxicity, and other apoptotic pathways coupled with ER stress may be involved in MeHg-induced cell death.

DOI： 10.2131/jts.49.55

Transcriptome analysis in various cell lines exposed to nitric oxide

Tohta Mizushima, Sho Kubota, Yuta Iijima, Nobumasa Takasugi, Takashi Uehara

The Journal of Toxicological Sciences 49 ( 6 ) 281 - 288 2024年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Toxicology

Nitric oxide (NO) plays a physiological role in signal transduction and excess or chronic NO has toxic effects as an inflammatory mediator. NO reversibly forms protein S-nitrosylation and exerts toxicological functions related to disease progression. DNA methyltransferases, epigenome-related enzymes, are inhibited in enzymatic activity by S-nitrosylation. Therefore, excess or chronic NO exposure may cause disease by altering gene expression. However, the effects of chronic NO exposure on transcriptome are poorly understood. Here, we performed transcriptome analysis of A549, AGS, HEK293T, and SW48 cells exposed to NO (100 μM) for 48 hr. We showed that the differentially expressed genes were cell-specific. Gene ontology analysis showed that the functional signature of differentially expressed genes related to cell adhesion or migration was upregulated in several cell lines. Gene set enrichment analysis indicated that NO stimulated inflammation-related gene expression in various cell lines. This finding supports previous studies showing that NO is closely involved in inflammatory diseases. Overall, this study elucidates the pathogenesis of NO-associated inflammatory diseases by focusing on changes in gene expression.

DOI： 10.2131/jts.49.281

Attenuation of protein arginine dimethylation via S-nitrosylation of protein arginine methyltransferase 1

Rikako Taniguchi, Yuto Moriya, Naoshi Dohmae, Takehiro Suzuki, Kengo Nakahara, Sho Kubota, Nobumasa Takasugi, Takashi Uehara

Journal of Pharmacological Sciences 154 ( 3 ) 209 - 217 2023年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier BV

Upregulation of nitric oxide (NO) production contributes to the pathogenesis of numerous diseases via S-nitrosylation, a post-translational modification of proteins. This process occurs due to the oxidative reaction between NO and a cysteine thiol group; however, the extent of this reaction remains unknown. S-Nitrosylation of PRMT1, a major asymmetric arginine methyltransferase of histones and numerous RNA metabolic proteins, was induced by NO donor treatment. We found that nitrosative stress leads to S-nitrosylation of cysteine 119, located near the active site, and attenuates the enzymatic activity of PRMT1. Interestingly, RNA sequencing analysis revealed similarities in the changes in expression elicited by NO and PRMT1 inhibitors or knockdown. A comprehensive search for PRMT1 substrates using the proximity-dependent biotin identification method highlighted many known and new substrates, including RNA-metabolizing enzymes. To validate this result, we selected the RNA helicase DDX3 and demonstrated that arginine methylation of DDX3 is induced by PRMT1 and attenuated by NO treatment. Our results suggest the existence of a novel regulatory system associated with transcription and RNA metabolism via protein S-nitrosylation.

DOI： 10.1016/j.jphs.2023.12.012

The Pursuit of the “Inside” of the Amyloid Hypothesis—Is C99 a Promising Therapeutic Target for Alzheimer’s Disease?

Nobumasa Takasugi, Masato Komai, Nanaka Kaneshiro, Atsuya Ikeda, Yuji Kamikubo, Takashi Uehara

Cells 12 ( 3 ) 454 - 454 2023年1月

掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer’s disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-β (Aβ), which accumulates in the patient brain. The “amyloid hypothesis” based therapy that places the aggregation and toxicity of Aβ at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. Aβ is produced from a type-I transmembrane protein, Aβ precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called βCTF), is a direct precursor of Aβ and accumulates in the AD patient’s brain to demonstrate toxicity independent of Aβ. Conventional drug discovery strategies have focused on Aβ toxicity on the “outside” of the neuron, but C99 accumulation might explain the toxicity on the “inside” of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and Aβ is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis.

DOI： 10.3390/cells12030454

Alterations in UPR Signaling by Methylmercury Trigger Neuronal Cell Death in the Mouse Brain 査読国際誌

Ryosuke Nomura, Nobumasa Takasugi, Hideki Hiraoka, Yuta Iijima, Takao Iwawaki, Yoshito Kumagai, Masatake Fujimura, Takashi Uehara

International Journal of Molecular Sciences 23 ( 23 ) 15412 - 15412 2022年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures specific areas of the brain. MeHg is known to induce oxidative and endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway has a dual nature in that it regulates and protects cells from an overload of improperly folded proteins in the ER, whereas excessively stressed cells are eliminated by apoptosis. Oxidative stress/ER stress induced by methylmercury exposure may tilt the UPR toward apoptosis, but there is little in vivo evidence of a direct link to actual neuronal cell death. Here, by using the ER stress-activated indicator (ERAI) system, we investigated the time course signaling alterations of UPR in vivo in the most affected areas, the somatosensory cortex and striatum. In the ERAI-Venus transgenic mice exposed to MeHg (30 or 50 ppm in drinking water), the ERAI signal, which indicates the activation of the cytoprotective pathway of the UPR, was only transiently enhanced, whereas the apoptotic pathway of the UPR was persistently enhanced. Furthermore, detailed analysis following the time course showed that MeHg-induced apoptosis is strongly associated with alterations in UPR signaling. Our results suggest that UPR modulation could be a therapeutic target for treating neuropathy.

DOI： 10.3390/ijms232315412

Lipid flippase dysfunction as a therapeutic target for endosomal anomalies in Alzheimer's disease. 査読国際誌

Nanaka Kaneshiro, Masato Komai, Ryosuke Imaoka, Atsuya Ikeda, Yuji Kamikubo, Takashi Saito, Takaomi C Saido, Taisuke Tomita, Tadafumi Hashimoto, Takeshi Iwatsubo, Takashi Sakurai, Takashi Uehara, Nobumasa Takasugi

iScience 25 ( 3 ) 103869 - 103869 2022年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer'| disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported that βCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between βCTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and AppNL-G-F/NL-G-F model mice. Furthermore, the T-RAP peptide derived from the βCTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD.

DOI： 10.1016/j.isci.2022.103869

Covalent N-arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor. 査読

Nakahara, K, Hamada, K, Tsuchida, T, Takasugi, N, Abiko, Y, Shien, K, Toyooka, S, Kumagai, Y, Uehara, T

J. Biol. Chem. 296 100524 2021年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The epidermal growth factor receptor (EGFR) is the most intensively investigated receptor tyrosine kinase. Several EGFR mutations and modifications have been shown to lead to abnormal self-activation, which plays a critical role in carcinogenesis. Environmental air pollutants, which are associated with cancer and respiratory diseases, can also activate EGFR. Specifically, the environmental electrophile 1,2- naphthoquinone (1,2-NQ), a component of diesel exhaust particles and particulate matter more generally, has previously been shown to impact EGFR signaling. However, the detailed mechanism of 1,2-NQ function is unknown. Here, we demonstrate that 1,2-NQ is a novel chemical activator of EGFR but not other EGFR family proteins. We found that 1,2-NQ forms a covalent bond, in a reaction referred to as N-arylation, with Lys80, which is in the ligand-binding domain. This modification activates the EGFR-Akt signaling pathway, which inhibits serum deprivation-induced cell death in a human lung adenocarcinoma cell line. Our study reveals a novel mode of EGFR pathway activation and suggests a link between abnormal EGFR activation and environmental pollutant- associated diseases such as cancer.

DOI： 10.1016/j.jbc.2021.100524

Spatiotemporal analysis of the UPR transition induced by methylmercury in the mouse brain. 査読国際誌

Hiraoka, H, Nomura, R, Takasugi, N, Akai, R, Iwawaki, T, Kumagai, Y, Fujimura, M, Uehara, T

Arch. Toxicol. 95 ( 4 ) 1241 - 1250 2021年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures a specific area of the brain. MeHg-mediated neurotoxicity is believed to be caused by oxidative stress and endoplasmic reticulum (ER) stress but the mechanism by which those stresses lead to neuronal loss is unclear. Here, by utilizing the ER stress-activated indicator (ERAI) system, we investigated the signaling alterations in the unfolded protein response (UPR) prior to neuronal apoptosis in the mouse brain. In ERAI transgenic mice exposed to MeHg (25 mg/kg, S.C.), the ERAI signal, which indicates activation of the cytoprotective pathway of the UPR, was detected in the brain. Interestingly, detailed ex vivo analysis showed that the ERAI signal was localized predominantly in neurons. Time course analysis of MeHg exposure (30 ppm in drinking water) showed that whereas the ERAI signal was gradually attenuated at the late phase after increasing at the early phase, activation of the apoptotic pathway of the UPR was enhanced in proportion to the exposure time. These results suggest that MeHg induces not only ER stress but also neuronal cell death via a UPR shift. UPR modulation could be a therapeutic target for treating neuropathy caused by electrophiles similar to MeHg.

DOI： 10.1007/s00204-021-02982-9

1,2-ナフトキノンによるN-アリール化を介したEGFR活性化機構の解析

中原健吾, 土田知貴, 安孫子ユミ, 熊谷嘉人, 上原孝

日本薬理学会年会要旨集 94 2-Y-E1-3 2021年

記述言語：日本語出版者・発行元：公益社団法人日本薬理学会

[Background] The epidermal growth factor receptor (EGFR) is the most intensively investigated receptor tyrosine kinase whose mutation or modification leads to abnormal self-activation, which plays a critical role in carcinogenesis. Although environmental air pollutants, which are associated with cancer and respiratory diseases, can activate EGFR, the detailed mechanisms remain unknown. In this study, we addressed to clarify the mechanisms by which the air pollutant 1,2-naphthoquinone (1,2-NQ) disrupts EGFR-Akt signaling and promotes oncogenic effects.

[Methods] Phosphorylated forms of Akt, EGFR, IGF-IR, and Insulin receptor were detected by western blotting using specific antibodies. The modification sites in EGFR were identified by LC-MS/MS analysis. Cell viability was assessed by WST-8 assay.

[Results] We found that 1,2-NQ stimulates the phosphorylation of EGFR, but not IGF-IR and Insulin receptor in A549 cells. LC-MS/MS analysis revealed that 1,2-NQ forms chemical modification with EGFR at ligand-binding domain I, Lys80. In addition, phosphorylation of Akt was observed by treatment with 1,2-NQ in a concentration-dependent manner. Activation of Akt by 1,2-NQ was suppressed by pretreatment with tyrphostinA25, a specific EGFR tyrosine kinase inhibitor. Moreover, 1,2-NQ exerted the cytoprotective effect against for serum deprivation-induced cell death through the activation of EGFR-Akt signaling.

[Conclusions] These findings showed that 1,2-NQ directly activates EGFR-Akt signaling via chemical modification of EGFR.

DOI： 10.1254/jpssuppl.94.0_2-y-e1-3

S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein. 査読国際誌

Kana Fujikawa, Kengo Nakahara, Nobumasa Takasugi, Tadashi Nishiya, Akihiro Ito, Koji Uchida, Takashi Uehara

Biochemical and biophysical research communications 524 ( 4 ) 910 - 915 2020年4月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

DOI： 10.1016/j.bbrc.2020.02.011

MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites. 国際誌

Keisuke Takeda, Shun Nagashima, Isshin Shiiba, Aoi Uda, Takeshi Tokuyama, Naoki Ito, Toshifumi Fukuda, Nobuko Matsushita, Satoshi Ishido, Takao Iwawaki, Takashi Uehara, Ryoko Inatome, Shigeru Yanagi

The EMBO journal 38 ( 15 ) e100999 2019年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

DOI： 10.15252/embj.2018100999

The Emerging Role of Electrophiles as a Key Regulator for Endoplasmic Reticulum (ER) Stress. 国際誌

Nobumasa Takasugi, Hideki Hiraoka, Kengo Nakahara, Shiori Akiyama, Kana Fujikawa, Ryosuke Nomura, Moeka Furuichi, Takashi Uehara

International journal of molecular sciences 20 ( 7 ) 2019年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

The unfolded protein response (UPR) is activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which is called ER stress. ER stress sensors PERK, IRE1, and ATF6 play a central role in the initiation and regulation of the UPR; they inhibit novel protein synthesis and upregulate ER chaperones, such as protein disulfide isomerase, to remove unfolded proteins. However, when recovery from ER stress is difficult, the UPR pathway is activated to eliminate unhealthy cells. This signaling transition is the key event of many human diseases. However, the precise mechanisms are largely unknown. Intriguingly, reactive electrophilic species (RES), which exist in the environment or are produced through cellular metabolism, have been identified as a key player of this transition. In this review, we focused on the function of representative RES: nitric oxide (NO) as a gaseous RES, 4-hydroxynonenal (HNE) as a lipid RES, and methylmercury (MeHg) as an environmental organic compound RES, to outline the relationship between ER stress and RES. Modulation by RES might be a target for the development of next-generation therapy for ER stress-associated diseases.

DOI： 10.3390/ijms20071783

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