| 1. |
Akiko Ogawa, Seiya Ohira, Yuri Kato, Tatsuya Ikuta, Shota Yanagida, Xinya Mi, Yukina Ishii, Yasunari Kanda, Motohiro Nishida, Asuka Inoue, Fan-Yan Wei, Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction., Communications biology, 10.1038/s42003-023-04888-x, 6, 1, 511-511, 2023.05, Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future.. |
| 2. |
Kazuhiro Nishiyama, Kohei Ariyoshi, Akiyuki Nishimura, Yuri Kato, Xinya Mi, Hitoshi Kurose, Sang Geon Kim, Motohiro Nishida, Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis., International journal of molecular sciences, 10.3390/ijms24043800, 24, 4, 2023.02, Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.. |
| 3. |
Xiaokang Tang, Akiyuki Nishimura, Kohei Ariyoshi, Kazuhiro Nishiyama, Yuri Kato, Elena A Vasileva, Natalia P Mishchenko, Sergey A Fedoreyev, Valentin A Stonik, Hyoung-Kyu Kim, Jin Han, Yasunari Kanda, Keitaro Umezawa, Yasuteru Urano, Takaaki Akaike, Motohiro Nishida, Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice., Marine drugs, 10.3390/md21010052, 21, 1, 2023.01, Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.. |
| 4. |
Yuri Kato, Kazuhiro Nishiyama, Jae Man Lee, Yuko Ibuki, Yumiko Imai, Takamasa Noda, Noriho Kamiya, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation., International journal of molecular sciences, 10.3390/ijms24010102, 24, 1, 2022.12, Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.. |
| 5. |
Sayaka Oda, Kazuhiro Nishiyama, Yuka Furumoto, Yohei Yamaguchi, Akiyuki Nishimura, Xiaokang Tang, Yuri Kato, Takuro Numaga-Tomita, Toshiyuki Kaneko, Supachoke Mangmool, Takuya Kuroda, Reishin Okubo, Makoto Sanbo, Masumi Hirabayashi, Yoji Sato, Yasuaki Nakagawa, Koichiro Kuwahara, Ryu Nagata, Gentaro Iribe, Yasuo Mori, Motohiro Nishida, Myocardial TRPC6-mediated Zn2+ influx induces beneficial positive inotropy through β-adrenoceptors., Nature communications, 10.1038/s41467-022-34194-9, 13, 1, 6374-6374, 2022.10, Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates β-adrenoceptor (βAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated βAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting β-arrestin-mediated βAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure.. |
| 6. |
Takuro Numaga-Tomita, Tsukasa Shimauchi, Yuri Kato, Kazuhiro Nishiyama, Akiyuki Nishimura, Kosuke Sakata, Hiroyuki Inada, Satomi Kita, Takahiro Iwamoto, Junichi Nabekura, Lutz Birnbaumer, Yasuo Mori, Motohiro Nishida, Inhibition of transient receptor potential cation channel 6 promotes capillary arterialization during post-ischaemic blood flow recovery., British journal of pharmacology, 10.1111/bph.15942, 2022.09, BACKGROUND AND PURPOSE: Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. EXPERIMENTAL APPROACH: Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. KEY RESULTS: Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. CONCLUSION AND IMPLICATIONS: Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation.. |
| 7. |
Yuri Kato, Kazuhiro Nishiyama, Akiyuki Nishimura, Takamasa Noda, Kaori Okabe, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, Drug repurposing for the treatment of COVID-19., Journal of pharmacological sciences, 10.1016/j.jphs.2022.04.007, 149, 3, 108-114, 2022.07, Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure.. |
| 8. |
Yuri Kato, Kengo Ohsugi, Yuto Fukuno, Ken Iwatsuki, Yuika Harada, Takaaki Miyaji, Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment., Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.2122158119, 119, 30, e2122158119, 2022.07, Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.. |
| 9. |
Tsukasa Shimauchi, Takuro Numaga-Tomita, Yuri Kato, Hiroyuki Morimoto, Kosuke Sakata, Ryosuke Matsukane, Akiyuki Nishimura, Kazuhiro Nishiyama, Atsushi Shibuta, Yutoku Horiuchi, Hitoshi Kurose, Sang Geon Kim, Yasuteru Urano, Takashi Ohshima, Motohiro Nishida, A TRPC3/6 Channel Inhibitor Promotes Arteriogenesis after Hind-Limb Ischemia., Cells, 10.3390/cells11132041, 11, 13, 2022.06, Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels.. |
| 10. |
Akiyuki Nishimura, Tomohiro Tanaka, Yuri Kato, Kazuhiro Nishiyama, Motohiro Nishida, Cardiac robustness regulated by reactive sulfur species., Journal of clinical biochemistry and nutrition, 10.3164/jcbn.21-84, 70, 1, 1-6, 2022.01, The human myocardium contains robust cells that constantly beat from birth to death without being replaced, even when exposed to various environmental stresses. Myocardial robustness is thought to depend primarily on the strength of the reducing power to protect the heart from oxidative stress. Myocardial antioxidant systems are controlled by redox reactions, primarily via the redox reaction of Cys sulfhydryl groups, such as found in thioredoxin and glutathione. However, the specific molecular entities that regulate myocardial reducing power have long been debated. Recently, reactive sulfide species, with excellent electron transfer ability, consisting of a series of multiple sulfur atoms, i.e., Cys persulfide and Cys polysulfides, have been found to play an essential role in maintaining mitochondrial quality and function, as well as myocardial robustness. This review presents the latest findings on the molecular mechanisms underlying mitochondrial energy metabolism and the maintenance of quality control by reactive sulfide species and provides a new insight for the prevention of chronic heart failure.. |
| 11. |
Kazuhiro Nishiyama, Akiyuki Nishimura, Kakeru Shimoda, Tomohiro Tanaka, Yuri Kato, Takahiro Shibata, Hiroshi Tanaka, Hitoshi Kurose, Yasu-Taka Azuma, Hideshi Ihara, Yoshito Kumagai, Takaaki Akaike, Philip Eaton, Koji Uchida, Motohiro Nishida, Redox-dependent internalization of the purinergic P2Y6 receptor limits colitis progression., Science signaling, 10.1126/scisignal.abj0644, 15, 716, eabj0644, 2022.01, After ligand stimulation, many G protein–coupled receptors (GPCRs) undergo β-arrestin–dependent desensitization, during which they are internalized and either degraded or recycled to the plasma membrane. Some GPCRs are not subject to this type of desensitization because they lack the residues required to interact with β-arrestins. We identified a mechanism of redox-dependent alternative internalization (REDAI) that promotes the internalization and degradation of the purinergic P2Y6 receptor (P2Y6R). Synthetic and natural compounds containing electrophilic isothiocyanate groups covalently modified P2Y6R at Cys220, which promoted the ubiquitylation of Lys137 and receptor internalization and degradation in various mouse and human cultured cell lines. Endogenous electrophiles also promoted ligand-dependent P2Y6R internalization and degradation. P2Y6R is highly abundant in inflammatory cells and promotes the pathogenesis of colitis. Deficiency in P2Y6R protected mice against experimentally induced colitis, and mice expressing a form of P2Y6R in which Cys220 was mutated to nonmodifiable serine were more sensitive to the induction of colitis. Several other GPCRs, including A2BAR, contain cysteine and lysine residues at the appropriate positions to mediate REDAI, and isothiocyanate stimulated the internalization of A2BAR and of a form of P2Y2R with insertions of the appropriate residues. Thus, endogenous and exogenous electrophiles may limit colitis progression through cysteine modification of P2Y6R and may also mediate internalization of other GPCRs.. |
| 12. |
Yuri Kato, Shigeru Yamada, Kazuhiro Nishiyama, Ayano Satsuka, Suyong Re, Daiki Tomokiyo, Jae Man Lee,Tomohiro Tanaka, Akiyuki Nishimura, Kenzo Yonemitsu, Hiroshi Asakura, Yuko Ibuki, Yumiko Imai, Noriho Kamiya, Kenji Mizuguchi, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry, bioRxiv preprint, https://doi.org/10.1101/2021.03.13.435221, 2021.03. |
| 13. |
Kakeru Shimoda, Akiyuki Nishimura, Caroline Sunggip, Tomoya Ito, Kazuhiro Nishiyama, Yuri Kato, Tomohiro Tanaka, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Motohiro Nishida, Modulation of P2Y6R expression exacerbates pressure overload-induced cardiac remodeling in mice., Scientific reports, 10.1038/s41598-020-70956-5, 10, 1, 13926-13926, 2020.08, Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y6 receptor (P2Y6R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y6R prevents or promotes heart failure. We demonstrate that inhibition of P2Y6R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y6R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y6R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y6R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y6R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y6R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y6R exacerbates pressure overload-induced heart failure in mice, although P2Y6R in cardiomyocytes contributes to the progression of cardiac fibrosis.. |
| 14. |
Jing Che, Naoki Yamaji, Takaaki Miyaji, Namiki Mitani-Ueno, Yuri Kato, Ren Fang Shen, Jian Feng Ma, Node-Localized Transporters of Phosphorus Essential for Seed Development in Rice., Plant & cell physiology, 10.1093/pcp/pcaa074, 61, 8, 1387-1398, 2020.08, About 60-85% of total phosphorus (P) in cereal crops is finally allocated to seeds, where it is required for seed development, germination and early growth. However, little is known about the molecular mechanisms underlying P allocation to seeds. Here, we found that two members (OsPHO1;1 and OsPHO1;2) of the PHO1 gene family are involved in the distribution of P to seeds in rice. Both OsPHO1;1 and OsPHO1;2 were localized to the plasma membrane and showed influx transport activities for inorganic phosphate. At the reproductive stage, both OsPHO1;1 and OsPHO1;2 showed higher expression in node I, the uppermost node connecting to the panicle. OsPHO1;1 was mainly localized at the phloem region of diffuse vascular bundles (DVBs) of node I, while OsPHO1;2 was expressed in the xylem parenchyma cells of the enlarged vascular bundles (EVBs). In addition, they were also expressed in the ovular vascular trace, the outer layer of the inner integument (OsPHO1;1) and in the nucellar epidermis (OsPHO1;2) of caryopses. Knockout of OsPHO1;2, as well as OsPHO1;1 to a lesser extent, decreased the distribution of P to the seed, resulting in decreased seed size and delayed germination. Taken together, OsPHO1;2 expressed in node I is responsible for the unloading of P from the xylem of EVBs, while OsPHO1;1 is involved in reloading P into the phloem of DVBs for subsequent allocation of P to seeds. Furthermore, OsPHO1;1 and OsPHO1;2 expression in the caryopsis is important for delivering P from the maternal tissues to the filial tissues for seed development.. |
| 15. |
Mamiyo Kawakami, Narinobu Juge, Yuri Kato, Hiroshi Omote, Yoshinori Moriyama, Takaaki Miyaji, Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli, Journal of Proteome Research, 10.1021/acs.jproteome.7b00777, 17, 3, 1108-1119, 2018.03, Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.. |
| 16. |
Yuri Kato, Miki Hiasa, Reiko Ichikawa, Nao Hasuzawa, Atsushi Kadowaki, Ken Iwatsuki, Kazuhiro Shima, Yasuo Endo, Yoshiro Kitahara, Tsuyoshi Inoue, Masatoshi Nomura, Hiroshi Omote, Yoshinori Moriyama, Takaaki Miyaji, Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1704847114, 114, 31, E6297-E6305, 2017.08, Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl-. A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT-/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.. |
| 17. |
Yuri Kato, Hiroshi Omote, Takaaki Miyaji, Inhibitors of ATP release inhibit vesicular nucleotide transporter, Biological and Pharmaceutical Bulletin, 10.1248/bpb.b13-00544, 36, 11, 1688-1691, 2013.11, Vesicular nucleotide transporter (VNUT) is responsible for vesicular ATP storage in ATP-secreting cells. In the present study, we examined the effects on VNUT-mediated transport of ATP release inhibitors such as ATP-binding cassette (ABC) proteins, hemichannels, maxi anion channels and P2X7 receptor. The ATP transport activity of proteoliposomes containing purified human VNUT was blocked by glibenclamide, carbenoxolone, 18 á-glycyrrhetinic acid, flufenamic acid, arachidonic acid and A438079 without the formation of Äø (positive inside) as a driving force being affected. Thus, inhibitors of ATP release may inhibit VNUT and subsequent ATP release, since the previous works proved that inhibitors of ATP release blocked VNUT-mediated ATP release at the cell level. © 2013 The Pharmaceutical Society of Japan.. |
