研究等業績 - 原著論文 - 張 田力
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Supersulfides suppress type-Ⅰ and type-Ⅱ interferon responses by blocking JAK/STAT signaling in macrophages.
Xiaoyan Li, Touya Toyomoto, Tianli Zhang, Chunyu Guo, Stephen Lindahl, Hiroyasu Tsutsuki, Ming Xian, Tomohiro Sawa
International immunology 2024年06月
研究論文（学術雑誌）
Interferons (IFNs) are cytokines produced and secreted by immune cells when viruses, tumor cells, and so forth, invade the body. Their biological effects are diverse, including antiviral, cell growth-inhibiting, and antitumor effects. The main subclasses of interferons include type-I (e.g., IFN-α and IFN-β) and type-II (IFN-γ), which activate intracellular signals by binding to type-I and type-II IFN receptors, respectively. We have previously shown that when macrophages are treated with supersulfide donors, which have polysulfide structures in which three or more sulfur atoms are linked within the molecules, IFN-β-induced cellular responses, including signal transducer and activator of transcription 1 (STAT1) phosphorylation and inducible nitric oxide synthase (iNOS) expression, were strongly suppressed. However, the subfamily specificity of the suppression of IFN signals by supersulfides and the mechanism of this suppression are unknown. This study demonstrated that supersulfide donor N-acetyl-L-cysteine tetrasulfide (NAC-S2) can inhibit IFN signaling in macrophages stimulated not only with IFN-α/β but also with IFN-γ. Our data suggest that NAC-S2 blocks phosphorylation of Janus kinases (JAKs), thereby contributes to the inhibition of phosphorylation of STAT1. Under the current experimental conditions, hydrogen sulfide (H2S) donor NaHS failed to inhibit IFN signaling. Similar to NAC-S2, carbohydrate-based supersulfide donor thioglucose tetrasulfide (TGS4) was capable of strongly inhibiting tumor necrosis factor-αproduction, iNOS expression, and nitric oxide production from macrophages stimulated with lipopolysaccharide. Further understanding of molecular mechanisms how supersulfide donors exhibit their inhibitory actions towards JAK/STAT signaling is necessary basis for development of supersulfide-based therapeutic strategy against autoimmune disorders with dysregulated IFN signaling.
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Regulation of innate immune and inflammatory responses by supersulfides.
Hiroyasu Tsutsuki, Tianli Zhang, Takaaki Akaike, Tomohiro Sawa
International immunology ( Oxford University Press (OUP) ) 36 ( 4 ) 143 - 154 2024年01月 [査読有り]
研究論文（学術雑誌）
Innate immunity plays an important role in host defense against microbial infections. It also participates in activation of acquired immunity through cytokine production and antigen presentation. Pattern recognition receptors such as Toll-like receptors and nucleotide oligomerization domain-like receptors sense invading pathogens and associated tissue injury, after which inflammatory mediators such as pro-inflammatory cytokines and nitric oxide are induced. Supersulfides are molecular species possessing catenated sulfur atoms such as persulfide and polysulfide moieties. They have recently been recognized as important regulators in cellular redox homeostasis by acting as potent antioxidants and nucleophiles. In addition, recent studies suggested that supersulfides are critically involved in the regulation of innate immune and inflammatory responses. In this review, we summarize current knowledge of the chemistry and biology of supersulfides, with particular attention to their roles in regulation of innate immune and inflammatory responses. Studies with animal models of infection and inflammation demonstrated the potent anti-inflammatory functions of supersulfides such as blocking pro-inflammatory signaling cascades, reducing oxidative stresses, and inhibiting replication of microbial pathogens including severe acute respiratory syndrome coronavirus 2. Precise understanding of how supersulfides regulate innate immune responses is the necessary requirement for developing supersulfide-based diagnostic as well as therapeutic strategies against inflammatory disorders.
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Thioglucose-derived tetrasulfide, a unique polysulfide model compound.
Stephen Lindahl, Meg Shieh, Tianli Zhang, Chunyu Guo, Jerome R Robinson, Tomohiro Sawa, Ming Xian
Redox biology 70 103045 - 103045 2024年01月 [査読有り]
研究論文（学術雑誌）
Polysulfides have received increased interest in redox biology due to their role as the precursors of H2S and persulfides. However, the compounds that are suitable for biological investigations are limited to cysteine- and glutathione-derived polysulfides. In this work, we report the preparation and evaluation of a novel polysulfide derived from thioglucose, which represents the first carbohydrate-based polysulfide. This compound, thioglucose tetrasulfide (TGS4), showed excellent stability and water solubility. H2S and persulfide production from TGS4, as well as its associated antioxidative property were also demonstrated. Additionally, TGS4 was demonstrated to significantly induce cellular sulfane sulfur level increase, in particular for the formation of hydropersulfides/trisulfides. These results suggest that TGS4 is a useful tool for polysulfide research.
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腸管出血性大腸菌毒素SubABの病原性発現機構に関わるレドックスバイオロジー
津々木 博康, 張 田力, 澤 智裕
薬学雑誌 ( 公益社団法人 日本薬学会 ) 144 ( 1 ) 57 - 60 2024年01月
研究論文（学術雑誌）
<p>AB<sub>5</sub> toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic <i>Escherichia coli</i> (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB<sub>5</sub> toxins, this indicates the involvement of redox regulation <i>via</i> chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.</p>
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Waliul Islam, Hiroyasu Tsutsuki, Azizur Rahman, Ayaka Harada, Tianli Zhang, Katsuhiko Ono, Rayhanul Islam, Foysal Hossen, Takuro Niidome, Tomohiro Sawa, Jun Fang
ACS Applied Polymer Materials ( American Chemical Society (ACS) ) 5 ( 12 ) 10289 - 10302 2023年11月 [査読有り]
研究論文（学術雑誌）
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Yurika Tahara, Mikako Fujita, Tianli Zhang, Dongxing Wang, Hiroshi Tateishi, Akihiro Togami, Perpetual Nyame, Hiromi Terasawa, Nami Monde, Joyce Appiah-Kubi, Wright Ofotsu Amesimeku, Doaa Husham Majeed Alsaadi, Mikiyo Wada, Koji Sugimura, Sevgi Gezici, Halilibrahim Ciftci, Faruk Karahan, Nazim Sekeroglu, Masami Otsuka, Tomohiro Sawa, Yosuke Maeda, Takashi Watanabe, Kazuaki Monde
Biological and Pharmaceutical Bulletin ( Pharmaceutical Society of Japan ) 46 ( 11 ) 1535 - 1547 2023年11月 [査読有り]
研究論文（学術雑誌）
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Touya Toyomoto, Katsuhiko Ono, Tomoo Shiba, Kenta Momitani, Tianli Zhang, Hiroyasu Tsutsuki, Takeshi Ishikawa, Kanae Hoso, Koma Hamada, Azizur Rahman, Liping Wen, Yosuke Maeda, Keiichi Yamamoto, Masao Matsuoka, Kenjiro Hanaoka, Takuro Niidome, Takaaki Akaike, Tomohiro Sawa
Frontiers in Microbiology ( Frontiers Media SA ) 14 2023年10月 [査読有り]
研究論文（学術雑誌）
A principal concept in developing antibacterial agents with selective toxicity is blocking metabolic pathways that are critical for bacterial growth but that mammalian cells lack. Serine O-acetyltransferase (CysE) is an enzyme in many bacteria that catalyzes the first step in l-cysteine biosynthesis by transferring an acetyl group from acetyl coenzyme A (acetyl-CoA) to l-serine to form O-acetylserine. Because mammalian cells lack this l-cysteine biosynthesis pathway, developing an inhibitor of CysE has been thought to be a way to establish a new class of antibacterial agents. Here, we demonstrated that alkyl gallates such as octyl gallate (OGA) could act as potent CysE inhibitors in vitro and in bacteria. Mass spectrometry analyses indicated that OGA treatment markedly reduced intrabacterial levels of l-cysteine and its metabolites including glutathione and glutathione persulfide in Escherichia coli to a level similar to that found in E. coli lacking the cysE gene. Consistent with the reduction of those antioxidant molecules in bacteria, E. coli became vulnerable to hydrogen peroxide-mediated bacterial killing in the presence of OGA. More important, OGA treatment intensified susceptibilities of metallo-β-lactamase-expressing Gram-negative bacteria (E. coli and Klebsiella pneumoniae) to carbapenem. Structural analyses showed that alkyl gallate bound to the binding site for acetyl-CoA that limits access of acetyl-CoA to the active site. Our data thus suggest that CysE inhibitors may be used to treat infectious diseases caused by drug-resistant Gram-negative bacteria not only via direct antibacterial activity but also by enhancing therapeutic potentials of existing antibiotics.
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Disulfiram encapsulated in polymer nanoparticles ameliorates thioacetamide-induced liver injury
Wei Xu, Yuta Kadoya, Kaito Sennari, Waliul Islam, Tianli Zhang, Tomohiro Sawa, Fumika Akizuki, Hisaaki Hirose, Shiroh Futaki, Yukio Fujiwara, Yoshihiro Komohara, Takuro Niidome
Journal of Drug Delivery Science and Technology ( Elsevier BV ) 88 104981 - 104981 2023年10月 [査読有り]
研究論文（学術雑誌）
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Redox Regulation of Xenobiotics by Reactive Sulfur and Supersulfide Species.
Tianli Zhang, Takaaki Akaike, Tomohiro Sawa
Antioxidants & redox signaling ( Mary Ann Liebert Inc ) 40 ( 10-12 ) 679 - 690 2023年09月 [査読有り]
研究論文（学術雑誌）
Significance: Routine exposure to xenobiotics is unavoidable during our lifetimes. Certain xenobiotics are hazardous to human health, and are metabolized in the body to render them less toxic. During this process, several detoxification enzymes cooperatively metabolize xenobiotics. Glutathione (GSH) conjugation plays an important role in the metabolism of electrophilic xenobiotics. Recent Advances: Recent advances in reactive sulfur and supersulfide (RSS) analyses showed that persulfides and polysulfides bound to low-molecular-weight thiols, such as GSH, and to protein thiols are abundant in both eukaryotes and prokaryotes. The highly nucleophilic nature of hydropersulfides and hydropolysulfides contributes to cell protection against oxidative stress and electrophilic stress. Critical Issues: In contrast to GSH conjugation to electrophiles that is aided by glutathione S-transferase (GST), persulfides and polysulfides can directly form conjugates with electrophiles without the catalytic actions of GST. The polysulfur bonds in the conjugates are further reduced by perthioanions and polythioanions derived from RSS to form sulfhydrated metabolites that are no longer electrophilic but rather nucleophilic, and differ from metabolites that are formed via GSH conjugation. Future Directions: In view of the abundance of RSS in cells and tissues, metabolism of xenobiotics that is mediated by RSS warrants additional investigations, such as studies of the impact of microbiota-derived RSS on xenobiotic metabolism. Metabolites formed from reactions between electrophiles and RSS may be potential biomarkers for monitoring exposure to electrophiles and for studying their metabolism by RSS.
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Non-canonical inflammasome activation analysis in a mouse model of Citrobacter rodentium infection.
Hiroyasu Tsutsuki, Tianli Zhang, Kinnosuke Yahiro, Touya Toyomoto, Tomohiro Sawa
STAR protocols 3 ( 4 ) 101741 - 101741 2022年12月 [査読有り]
研究論文（学術雑誌）
Infection of mice with Citrobacter rodentium is a useful model for studying the pathogenicity of enteropathogenic and enterohemorrhagic Escherichia coli, pathogens that have a close association with humans. Here, we provide a protocol detailing the approaches for non-canonical inflammasome analysis in a mouse model of C. rodentium infection, including preparation of bacteria, oral administration of bacteria to mice, counting colony-forming units to quantify bacterial colonization, and analysis of expression and activation of inflammasome-related factors. For complete details on the use and execution of this protocol, please refer to Tsutsuki et al. (2022).
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A potential model of systemic sclerosis with pulmonary hypertension induced by monocrotaline plus bleomycin.
Xia Fang, Chao He, Xudong Ni, Tianli Zhang, Qianyu Li, Yi Luo, Wei Long, Rui Wu
Clinical and experimental hypertension (New York, N.Y. : 1993) 44 ( 6 ) 507 - 513 2022年08月
研究論文（学術雑誌）
OBJECTIVE: The lack of a well-established animal model limits the clarification of the detailed mechanisms of the pathogenesis of systemic sclerosis with pulmonary hypertension (SSc-PH) and the development of effective treatments for it. METHODS: In this study, New Zealand rabbits were injected with monocrotaline (MCT), bleomycin (BLM), and MCT plus BLM, respectively. Three and six weeks after the first injection, the mean pulmonary artery pressure (mPAP) was measured. Skin and lung samples were isolated and the histological changes were analyzed by hematoxylin and eosin staining or Masson's trichrome staining. RESULTS: All groups of rabbits showed an increased mean mPAP compared with the saline-injected rabbits. The high mPAP persisted until week six only in the MCT and MCT + BLM groups. Furthermore, persistent high Fulton's indices were found in the MCT and MCT + BLM groups, indicating that these treatments successfully induced right ventricular hypertrophy. The rabbits in the MCT + BLM group developed severe lung inflammation, as evidenced by a high level of neutrophil infiltration in the pulmonary interstitium. Importantly, pathological changes of the skin in the MCT + BLM group were observed, and further damage to the skin was caused by additional exposure to MCT plus BLM. Meanwhile, an excessive production of cytokines, including tumor necrosis factor alpha (TNF-α), and transforming growth factor beta 1 (TGF-β1), were detected in the MCT + BLM group. CONCLUSION: These data indicate that SSc-PH induced by co-injection with MCT plus BLM shows persistent fibrosis and progressive PH, constituting a potential study model for SSc-PH.
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Ayaka Harada, Hiroyasu Tsutsuki, Tianli Zhang, Kinnosuke Yahiro, Tomohiro Sawa, Takuro Niidome
Nanomaterials (Basel, Switzerland) ( MDPI AG ) 12 ( 13 ) 2161 2022年06月 [査読有り]
研究論文（学術雑誌）
Advances in drug delivery systems (DDSs) have enabled the specific delivery of drugs to target cells. Subtilase cytotoxin (SubAB) produced by certain enterohemorrhagic Escherichia coli strains induces endoplasmic reticulum (ER) stress and suppresses nitric oxide generation in macrophages. We previously reported that modification of SubAB with poly(D,L-lactide-co-glycolic) acid (PLGA) nanoparticles (SubAB-PLGA NPs) increased intracellular uptake of SubAB and had an anti-inflammatory effect on macrophages. However, specific delivery of SubAB to macrophages could not be achieved because its effects on other cell types were not negligible. Therefore, to suppress non-specific SubAB binding, we used low-binding mutant SubABS35A (S35A) in which the 35th serine of the B subunit was mutated to alanine. In a macrophage cell line, PLGA NPs modified with S35A (S35A-PLGA NPs) induced ER stress and had anti-inflammatory effects similar to WT-PLGA NPs. However, in an epithelial cell line, S35A-PLGA NPs induced lower ER stress than WT-PLGA NPs. These results suggest that S35A is selectively delivered to macrophages rather than epithelial cells by modification with PLGA NPs and exerts anti-inflammatory effects. Our findings provide a useful technique for protein delivery to macrophages and encourage medical applications of DDSs for the treatment of inflammatory diseases.
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Subtilase cytotoxin from Shiga-toxigenic Escherichia coli impairs the inflammasome and exacerbates enteropathogenic bacterial infection.
Hiroyasu Tsutsuki, Tianli Zhang, Kinnosuke Yahiro, Katsuhiko Ono, Yukio Fujiwara, Sunao Iyoda, Fan-Yan Wei, Kazuaki Monde, Kazuko Seto, Makoto Ohnishi, Hiroyuki Oshiumi, Takaaki Akaike, Tomohiro Sawa
iScience 25 ( 4 ) 104050 - 104050 2022年04月 [査読有り]
研究論文（学術雑誌）
Subtilase cytotoxin (SubAB) is an AB5 toxin mainly produced by the locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli (STEC) strain such as O113:H21, yet the contribution of SubAB to STEC infectious disease is unclear. We found that SubAB reduced activation of the STEC O113:H21 infection-induced non-canonical NLRP3 inflammasome and interleukin (IL)-1β and IL-18 production in murine macrophages. Downstream of lipopolysaccharide signaling, SubAB suppressed caspase-11 expression by inhibiting interferon-β/STAT1 signaling, followed by disrupting formation of the NLRP3/caspase-1 assembly. These inhibitions were regulated by PERK/IRE1α-dependent endoplasmic reticulum (ER) stress signaling initiated by cleavage of the host ER chaperone BiP by SubAB. Our murine model of SubAB-producing Citrobacter rodentium demonstrated that SubAB promoted C. rodentium proliferation and worsened symptoms such as intestinal hyperplasia and diarrhea. These findings highlight the inhibitory effect of SubAB on the NLRP3 inflammasome via ER stress, which may be associated with STEC survival and infectious disease pathogenicity in hosts.
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New insights into the regulatory roles of glutathione in NLRP3-inflammasome-mediated immune and inflammatory responses.
Tianli Zhang, Hiroyasu Tsutsuki, Xiaoyan Li, Tomohiro Sawa
Journal of biochemistry 171 ( 4 ) 367 - 377 2022年03月 [査読有り]
研究論文（学術雑誌）
Glutathione (GSH) is the most abundant non-protein thiol (-SH) in mammalian cells. Its synthesis and metabolism serve to maintain cellular reduction-oxidation (redox) homeostasis, which is important for multiple cellular processes including proliferation, differentiation and death. An accumulating body of evidence suggests that the essential roles of GSH extended far beyond its oxidant and electrophile scavenger activities and regulatory role in the lifespan of cells. Recent findings revealed that altered GSH levels are closely associated with a wide range of pathologies including bacterial and viral infections, neurodegenerative diseases and autoimmune disorders, all of which are also characterized by aberrant activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. As a result of these findings, GSH was assigned a central role in influencing the activation of the NLRP3 inflammasome. To expand on our recent advances in understanding this process, we discuss here the emerging roles of GSH in activation of the NLRP3 inflammasome, and the therapeutic potential of GSH in its associated pathologies.
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Development of potent antipseudomonal β‐lactams by means of polycarboxylation of aminopenicillins
Shahinur Akter, Yohei Migiyama, Hiroyasu Tsutsuki, Katsuhiko Ono, Chika Hamasaki, Tianli Zhang, Kenki Miyao, Touya Toyomoto, Keiichi Yamamoto, Waliul Islam, Takuro Sakagami, Hirotaka Matsui, Yoshihiro Yamaguchi, Tomohiro Sawa
Microbiology and Immunology ( Wiley ) 65 ( 10 ) 449 - 461 2021年08月
研究論文（学術雑誌）
<jats:title>Abstract</jats:title><jats:p><jats:italic>Pseudomonas aeruginosa</jats:italic> is a Gram‐negative opportunistic pathogen that presents a serious risk to immunosuppressed individuals and other extremely vulnerable patients such as those in intensive care units. The emergence of multidrug‐resistant <jats:italic>Pseudomonas</jats:italic> strains has increased the need for new antipseudomonal agents. In this study, a series of amino group‐modified aminopenicillin derivatives was synthesized that have different numbers of carboxyl groups and structurally resemble carboxypenicillin–ureidopenicillin hybrids, and their antipseudomonal activities were evaluated. Among the derivatives synthesized, diethylenetriaminepentaacetic acid (DTPA)‐modified amoxicillin (DTPA‐Amox) showed potent antipseudomonal activity, not only against the laboratory strain PAO1 but also against clinically isolated <jats:italic>Pseudomonas</jats:italic> strains that were resistant to piperacillin and carbenicillin. DTPA‐Amox had no obvious cytotoxic effects on cultured mammalian cells. In addition, in an in vivo model of leukopenia, DTPA‐Amox treatment produced a moderate but statistically significant improvement in the survival of mice with <jats:italic>P. aeruginosa</jats:italic> strain PAO1 infection. These data suggest that polycarboxylation by DTPA conjugation is an effective approach to enhance antipseudomonal activity of aminopenicillins.</jats:p>
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ATP exposure stimulates glutathione efflux as a necessary switch for NLRP3 inflammasome activation.
Tianli Zhang, Hiroyasu Tsutsuki, Waliul Islam, Katsuhiko Ono, Kohsuke Takeda, Takaaki Akaike, Tomohiro Sawa
Redox biology 41 101930 - 101930 2021年05月 [査読有り]
研究論文（学術雑誌）
The NLRP3 inflammasome is a multiprotein complex responsible for the maturation of precursor forms of interleukin (IL)-1β and IL-18 into active proinflammatory cytokines. Increasing evidence suggests that modulation of redox homeostasis contributes to the activation of the NLRP3 inflammasome. However, specific mechanistic details remain unclear. We demonstrate here that ATP exposure evoked a sharp decrease in glutathione (GSH) levels in macrophages, which led to NLRP3 inflammasome activation. We detected an increase in GSH levels in culture supernatants that was comparable to the GSH decrease in macrophages, which suggests that exposure to ATP stimulated GSH efflux. Exogenous addition of P2X7 receptor antagonist, GSH, or the oxidized form GSSG attenuated this efflux. Also, exogenous GSH or GSSG strongly inhibited NLRP3 inflammasome activation in vitro and in vivo. These data suggest that GSH efflux controls NLRP3 inflammasome activation, which may lead to development of novel therapeutic strategies for NLRP3 inflammasome-associated disorders.
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Katsuhiko Ono, Yusuke Kitamura, Tianli Zhang, Hiroyasu Tsutsuki, Azizur Rahman, Toshihiro Ihara, Takaaki Akaike, Tomohiro Sawa
ACS Chemical Biology ( American Chemical Society (ACS) ) 16 ( 4 ) 731 - 739 2021年03月
研究論文（学術雑誌）
Hydrogen sulfide (H2S) formed during sulfur metabolism in bacteria has been implicated in the development of intrinsic resistance to antibacterial agents. Despite the conversion of H2S to hydropersulfides greatly enhancing the biochemical properties of H2S such as antioxidant activity, the effects of hydropersulfides on antibiotic resistance have remained unknown. In this work, we investigated the effects of H2S alone or together with cystine to form cysteine hydropersulfide (CysSSH) on the activities of antibacterial agents. By using the disc diffusion test, we found that CysSSH treatment effectively inactivated β-lactams of the penicillin class (penicillin G and ampicillin) and the carbapenem class (meropenem). These β-lactams were resistant to treatment with H2S alone or cystine alone. In contrast, cephalosporin class β-lactams (cefaclor and cefoperazone) and non-β-lactam antibiotics (tetracycline, kanamycin, erythromycin, and ofloxacin) were stable after CysSSH treatment. Chromatographic and mass spectrometric analyses revealed that CysSSH directly reacted with β-lactams to form β-lactam ring-opened carbothioic S-acids (BL-COSH). Furthermore, we demonstrated that certain bacteria (e.g., Escherichia coli and Staphylococcus aureus) efficiently decomposed β-lactam antibiotics to form BL-COSH, which were transported to the extracellular space. These data suggest that CysSSH-mediated β-lactam decomposition may contribute to intrinsic bacterial resistance to β-lactams. BL-COSH may become useful biomarkers for CysSSH-mediated β-lactam resistance and for investigation of potential antibacterial adjuvants that can enhance the antibacterial activity of β-lactams by reducing the hydropersulfides in bacteria.
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Fusobacterium nucleatum confers chemoresistance by modulating autophagy in oesophageal squamous cell carcinoma.
Yang Liu, Yoshifumi Baba, Takatsugu Ishimoto, Hiroyasu Tsutsuki, Tianli Zhang, Daichi Nomoto, Kazuo Okadome, Kensuke Yamamura, Kazuto Harada, Kojiro Eto, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yohei Nagai, Shiro Iwagami, Yuji Miyamoto, Naoya Yoshida, Yoshihiro Komohara, Masaki Ohmuraya, Xiaoming Wang, Jaffer A Ajani, Tomohiro Sawa, Hideo Baba
British journal of cancer 124 ( 5 ) 963 - 974 2021年03月 [査読有り]
研究論文（学術雑誌）
BACKGROUND: Fusobacterium nucleatum (F. nucleatum) is a gut microbe implicated in gastrointestinal tumorigenesis. Predicting the chemotherapeutic response is critical to developing personalised therapeutic strategies for oesophageal cancer patients. The present study investigated the relationship between F. nucleatum and chemotherapeutic resistance in oesophageal squamous cell carcinoma (ESCC). METHODS: We examined the relationship between F. nucleatum and chemotherapy response in 120 ESCC resected specimens and 30 pre-treatment biopsy specimens. In vitro studies using ESCC cell lines and co-culture assays further uncovered the mechanism underlying chemotherapeutic resistance. RESULTS: ESCC patients with F. nucleatum infection displayed lesser chemotherapeutic response. The infiltration and subsistence of F. nucleatum in the ESCC cells were observed by transmission electron microscopy and laser scanning confocal microscopy. We also observed that F. nucleatum modulates the endogenous LC3 and ATG7 expression, as well as autophagosome formation to induce chemoresistance against 5-FU, CDDP, and Docetaxel. ATG7 knockdown resulted in reversal of F. nucleatum-induced chemoresistance. In addition, immunohistochemical studies confirmed the correlation between F. nucleatum infection and ATG7 expression in 284 ESCC specimens. CONCLUSIONS: F. nucleatum confers chemoresistance to ESCC cells by modulating autophagy. These findings suggest that targeting F. nucleatum, during chemotherapy, could result in variable therapeutic outcomes for ESCC patients.
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Antioxidative and anti-inflammatory actions of reactive cysteine persulfides.
Tianli Zhang, Hiroyasu Tsutsuki, Katushiko Ono, Takaaki Akaike, Tomohiro Sawa
Journal of clinical biochemistry and nutrition 68 ( 1 ) 5 - 8 2021年01月 [査読有り]
研究論文（学術雑誌）
Cysteine persulfide (CysSSH) and polysulfides (CysS[S] n H, n>1) are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Recent advances in the development of analytical methods for detection and quantification of persulfides and polysulfides have revealed the biological presence, in both prokaryotes and eukaryotes, of persulfide/polysulfide in diverse forms such as CysSSH, glutathione persulfide and protein persulfides. Accumulating evidence has suggested that persulfide/polysulfide species may involve in a variety of biological events such as biosyntheses of sulfur-containing molecules, tRNA modification, regulation of redox-dependent signal transduction, mitochondrial energy metabolism via sulfur respiration, cytoprotection from oxidative stress via their antioxidant activities, and anti-inflammation against Toll-like receptor-mediated inflammatory responses. Development of chemical sulfur donors may facilitate further understanding of physiological and pathophysiological roles of persulfide/polysulfide species, including regulatory roles of these species in immune responses.
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Involvement of protein disulfide isomerase in subtilase cytotoxin-induced cell death in HeLa cells
Hiroyasu Tsutsuki, Tianli Zhang, Ayaka Harada, Azizur Rahman, Katsuhiko Ono, Kinnosuke Yahiro, Takuro Niidome, Tomohiro Sawa
Biochemical and Biophysical Research Communications ( Elsevier BV ) 525 ( 4 ) 1068 - 1073 2020年05月 [査読有り]
研究論文（学術雑誌）
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Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.
Ayaka Harada, Hiroyasu Tsutsuki, Tianli Zhang, Ruda Lee, Kinnosuke Yahiro, Tomohiro Sawa, Takuro Niidome
Chemical & pharmaceutical bulletin 68 ( 4 ) 363 - 368 2020年 [査読有り]
研究論文（学術雑誌）
Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.
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Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock.
Tianli Zhang, Katsuhiko Ono, Hiroyasu Tsutsuki, Hideshi Ihara, Waliul Islam, Takaaki Akaike, Tomohiro Sawa
Cell chemical biology 26 ( 5 ) 686 - 698 2019年05月 [査読有り]
研究論文（学術雑誌）
Cysteine persulfide and cysteine polysulfides are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Accumulating evidence has suggested that cysteine persulfides/polysulfides are abundant in prokaryotes and eukaryotes and play important roles in diverse biological processes such as antioxidant host defense and redox-dependent signal transduction. Here, we show that enhancement of cellular polysulfides by using polysulfide donors developed in this study resulted in marked inhibition of lipopolysaccharide (LPS)-initiated macrophage activation. Polysulfide donor treatment strongly suppressed LPS-induced pro-inflammatory responses in macrophages by inhibiting Toll-like receptor 4 (TLR4) signaling. Other TLR signaling stimulants-including zymosan A-TLR2 and poly(I:C)-TLR3-were also significantly suppressed by polysulfur donor treatment. Administration of polysulfide donors protected mice from lethal endotoxin shock. These data indicate that cellular polysulfides negatively regulate TLR4-mediated pro-inflammatory signaling and hence constitute a potential target for inflammatory disorders.
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Synthesis of Pegylated Manganese Protoporphyrin as a Catalase Mimic and Its Therapeutic Application to Acetaminophen-Induced Acute Liver Failure.
Tianli Zhang, Jun Fang, Hiroyasu Tsutsuki, Katsuhiko Ono, Waliul Islam, Tomohiro Sawa
Biological & pharmaceutical bulletin 42 ( 7 ) 1199 - 1206 2019年 [査読有り]
研究論文（学術雑誌）
Metalloporphyrin derivatives have been investigated for their therapeutic potential for oxidative stress-related diseases because of their scavenging of reactive oxygen species (ROS). Here, we describe the synthesis, physicochemical properties, and ROS-scavenging activities of one such derivative-polyethylene glycol (PEG)-conjugated manganese protoporphyrin (PEG-MnPP). Carboxyl groups of the protoporphyrin ring at the C6 and C7 positions were first conjugated with ethylenediamine to introduce amino groups into the protoporphyrin structure. The amino groups were then reacted with succinimidyl PEG, with an average molecular weight of 2000, to obtain pegylated protoporphyrin (PEG-PP). Manganese was chelated to the protoporphyrin ring by incubating PEG-PP and manganese acetate in methanol. Dynamic light scattering and fluorescent spectrometry analyses revealed that PEG-MnPP self-assembled into nanoparticles in aqueous media with an apparent diameter of 70 nm. PEG-MnPP effectively eliminated hydrogen peroxide from cell culture media and protected cultured mammalian cells from toxic insults induced by hydrogen peroxide exposure or by 6-hydroxydopamine treatment. Intravenous administration of PEG-MnPP to mice significantly suppressed acute liver failure that had been induced by acetaminophen overdose. These data warrant additional investigation to study the therapeutic potential of PEG-MnPP as a water-soluble metalloporphyrin-based catalase mimic for oxidative stress-associated diseases.
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Reactive Cysteine Persulphides: Occurrence, Biosynthesis, Antioxidant Activity, Methodologies, and Bacterial Persulphide Signalling.
Tomohiro Sawa, Katsuhiko Ono, Hiroyasu Tsutsuki, Tianli Zhang, Tomoaki Ida, Motohiro Nishida, Takaaki Akaike
Advances in microbial physiology 72 1 - 28 2018年 [査読有り]
研究論文（学術雑誌）
Cysteine hydropersulphide (CysSSH) is a cysteine derivative having one additional sulphur atom bound to a cysteinyl thiol group. Recent advances in the development of analytical methods for detection and quantification of persulphides and polysulphides have revealed the biological presence, in both prokaryotes and eukaryotes, of hydropersulphides in diverse forms such as CysSSH, homocysteine hydropersulphide, glutathione hydropersulphide, bacillithiol hydropersulphide, coenzyme A hydropersulphide, and protein hydropersulphides. Owing to the chemical reactivity of the persulphide moiety, biological systems utilize persulphides as important intermediates in the synthesis of various sulphur-containing biomolecules. Accumulating evidence has revealed another important feature of persulphides: their potent reducing activity, which implies that they are implicated in the regulation of redox signalling and antioxidant functions. In this chapter, we discuss the biological occurrence and possible biosynthetic mechanisms of CysSSH and related persulphides, and we include descriptions of recent advances in the analytical methods that have been used to detect and quantitate persulphide species. We also discuss the antioxidant activity of persulphide species that contributes to protecting cells from reactive oxygen species-associated damage, and we examine the signalling roles of CysSSH in bacteria.
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Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.
Katsuhiko Ono, Minkyung Jung, Tianli Zhang, Hiroyasu Tsutsuki, Hiroshi Sezaki, Hideshi Ihara, Fan-Yan Wei, Kazuhito Tomizawa, Takaaki Akaike, Tomohiro Sawa
Free radical biology & medicine 106 69 - 79 2017年05月 [査読有り]
研究論文（学術雑誌）
Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34S-labeled L-cysteine from O-acetyl-L-serine and 34S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur (34S) and nitrogen (15N) atoms was also achieved by performing enzyme reactions with 15N-labeled L-serine, acetyl-CoA, and 34S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34S-labeled N-acetyl-L-cysteine (NAC) by incubating 34S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology.
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Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.
Khandaker Ahtesham Ahmed, Tianli Zhang, Katsuhiko Ono, Hiroyasu Tsutsuki, Tomoaki Ida, Soichiro Akashi, Keishi Miyata, Yuichi Oike, Takaaki Akaike, Tomohiro Sawa
Biological & pharmaceutical bulletin 40 ( 3 ) 365 - 374 2017年03月 [査読有り]
研究論文（学術雑誌）
Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.
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Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation.
Hiroyasu Tsutsuki, Minkyung Jung, Tianli Zhang, Katsuhiko Ono, Tomoaki Ida, Kohei Kunieda, Hideshi Ihara, Takaaki Akaike, Tomohiro Sawa
Biochemical and biophysical research communications 478 ( 1 ) 7 - 11 2016年09月 [査読有り]
研究論文（学術雑誌）
8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections.
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JNK regulatory molecule G5PR induces IgG autoantibody-producing plasmablasts from peritoneal B1a cells.
Masahiro Kitabatake, Miho Soma, Tianli Zhang, Kazuhiko Kuwahara, Yoshimi Fukushima, Takuya Nojima, Daisuke Kitamura, Nobuo Sakaguchi
Journal of immunology (Baltimore, Md. : 1950) 194 ( 4 ) 1480 - 8 2015年02月 [査読有り]
研究論文（学術雑誌）
Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black × New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody-secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells.