リソゾーム膜に電子伝達系は存在するか?

金沢大学薬学部研究課題/領域番号:02857344, 研究期間(年度):1990出典：研究課題「リソゾーム膜に電子伝達系は存在するか?」課題番号02857344（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-02857344/）を加工して作

リソソームの崩壊機構とGTP結合蛋白質

金沢大学薬学部リソソーム指向性の塩基性薬物はリソソームに蓄積し顆粒内pHを上昇させるとともに空胞化を起こす。この塩基性薬物による効果をセルフリー系で確認する目的で単離リソソームを塩基で処理したところリソソームはATP依存的に崩壊した(塩基性薬物依存性リソソーム崩壊)。空胞形成機構として,リソソーム間や他の空胞系顆粒の融合が考えられた。そこでリソソーム膜融合機構の解析を行った。その過程でGTPγSで処理したサイトゾルによりリソソームが崩壊することをみいだした(GTPγS依存性リソソーム崩壊)。(1)塩基性薬物依存性リソゾーム崩壊はプロトンポンプ依存的に,リソゾーム内にプロトン付加型塩基の塩が高濃度に蓄積するために,リソゾーム内の浸透圧が上昇し崩壊するものと考えられた。このリソゾームの崩壊はサイトゾルの添加で顕著に抑えられた。この機構はサイトゾル因子はによりリソソーム内に塩基の蓄積が阻害された結果と考えられた。このサイトゾル因子ラット肝臓から単離されたグルココルチコイド受容体の核内移行促進因子と同じであることを明らかにした。(2)リソソーム膜融合機構について,融合によるリソソームの比重変化を指標とした。in vitroでのリソソーム間膜融合のアッセイ系を用いた、リソソーム間膜融合はサイトゾル及びATP要求性で,かつ温度依存性であった。この融合機構は,(a)サイトゾル因子の他,リソソーム膜蛋白質の関与がある。(b)細胞内小胞輸送の融合装置の普遍的なコンポーネントと考えられているNSFや,GTP結合蛋白質の関与はない。(c)IP_3受容体チャンネルからの動員による一過性のカルシウムイオン濃度の上昇が必要である,などが示唆された。(3)GTPγS依存性リソソーム崩壊は,(i)GTPγSによるサイトゾルのGTP結合蛋白質の活性化(ii)活性化GTP結合蛋白質によるATP要求性のリソソームの崩壊,の2段階の部分反応によっていることが考えられた。GTP結合蛋白質によって活性化される標的蛋白質としてPLA_2が示唆された。研究課題/領域番号:06807167, 研究期間(年度):1994出典：研究課題「リソソームの崩壊機構とGTP結合蛋白質」課題番号06807167（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-06807167/）を加工して作

リソソームのホモティピカルな膜融合機構の解析

金沢大学薬学部リソソームの主たる役割である様々な生体内高分子の分解の際,リソソームにタンパク質が移行する重要なステップは,基質タンパク質を含む膜小胞がリソソームと融合して,その内容物をリソソームへ受け渡すことである.そこで本研究ではホモティピカルなリソソーム間膜融合に注目し,そのin vitroのアツセイ系の確立を試みた.リソソーム膜融合の検出には,抗原抗体複合体形成を利用した.抗原としてFluorescein isothiocyanate(FITC)を西洋わさびペルオキシダーゼ(horseradish peroxidase:HRP)で標識したもの,抗体として抗FITC抗体を用いた.またリソソームへのリガンドの取り込み効率を上げるため,それぞれをアシアロフェチュインまたはガラクトースで修飾した.両リガンドを取り込んだリソソームの調製は,それぞれのリガンドを別々のマウスに静脈内投与した後,40分後に肝臓からパーコール密度勾配遠心法を用いた.このリガンドを取り込ませたリソソームを試験管の中でサイトゾル,ATP存在下,37℃でインキュベートした.その後,融合によって形成した抗原抗体複合体をprotein A-Sepharoseで回収し,そのHRP活性を発光法によって測定しリソソーム膜融合の指標とした。その結果,リソソームの膜融合はエンドソームやゴルジ体の膜融合反応と同じ温度依存性を示し20℃以下では起こらなかった.さらに,サイトゾル,ATP濃度依存性を示し,膜融合阻害剤として用いられているN-エチルマレイミドおよびGTγSに対する感受性も示した.また,リソソーム膜融合は,エンドソーム膜融合にはみられないGTP要求性を示した.ln vivo系においてリソソーム酵素のリソソームへの輸送の阻害や,リソソームの形態変化を起こすことが報告されているウォルトマニンやノコダゾールに対する感受性を検討した結果,リソソーム膜融合はウォルトマニンで阻害されたが,ノコダゾールでは阻害されなかった.従って,in vitroにおけるリソソーム膜融合には,PI3キナーゼが関与するが,細胞骨格は関与しない可能性が示唆された.研究課題/領域番号:09877434, 研究期間(年度):1997出典：研究課題「リソソームのホモティピカルな膜融合機構の解析 」課題番号09877434（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09877434/）を加工して作

Change in pharmacokinetics of mycophenolic acid as a function of age in rats and effect of coadministered amoxicillin/clavulanate

Changes of mycophenolic acid (MPA) pharmacokinetics with aging were investigated in rats. We also compared the effect of concomitant amoxicillin/clavulanate combination (CVA/AMPC) on the pharmacokinetics of MPA in 4-week-old and 12-week-old rats (the package insert of CVA/AMPC warns of possible interaction with MPA). Four-week-old rats showed a 1.4-fold higher total body clearance of MPA and a lower volume of distribution of MPA (65%), compared to the values in 12-week-old rats. However, the difference in MPA pharmacokinetics disappeared when enterohepatic circulation was eliminated by bile duct cannulation (BDC). Concomitant CVA/AMPC significantly reduced plasma MPA concentration in intact rats of both age groups, and the age-dependent difference of MPA pharmacokinetics was no longer apparent. The effect of CVA/AMPC was not seen in rats that had undergone BDC, suggesting that the drug-drug interaction can be attributed to inhibition of enterohepatic circulation by CVA/AMPC. These results indicate that the aging-related alteration of MPA pharmacokinetics is a consequence of immature enterohepatic circulation in 4-week-old rats. Higher doses of MPA may be necessary in juveniles. © 2012 The Pharmaceutical Society of Japan

Protective action of nipradilol mediated through S-nitrosylation of Keap1 and HO-1 induction in retinal ganglion cells

Nipradilol (Nip), which has α1- and β-adrenoceptor antagonist and nitric oxide (NO)-donating properties, has clinically been used as an anti-glaucomatous agent in Japan. NO mediates cellular signaling pathways that regulate physiological functions. The major signaling mechanisms mediated by NO are cGMP-dependent signaling and protein S-nitrosylation-dependent signalings. Nip has been described as having neuroprotective effects through cGMP-dependent pathway in retinal ganglion cells (RGCs). However, the effect seems to be partial. On the other hand, whether Nip can prevent cell death through S-nitrosylation is not yet clarified. In this study, we therefore focused on the neuroprotective mechanism of Nip through S-nitrosylation. Nip showed a dramatic neuroprotective effect against oxidative stress-induced death of RGC-5 cells. However, denitro-nipradilol, which does not have NO-donating properties, was not protective against oxidative stress. Furthermore, an NO scavenger significantly reversed the protective action of Nip against oxidative stress. In addition, we demonstrated that α1- or β-adrenoceptor antagonists (prazosin or timolol) did not show any neuroprotective effect against oxidative stress in RGC-5 cells. We also demonstrated that Nip induced the expression of the NO-dependent antioxidant enzyme, heme oxygenase-1 (HO-1). S-nitrosylation of Kelch-like ECH-associated protein by Nip was shown to contribute to the translocation of NF-E2-related factor 2 to the nucleus, and triggered transcriptional activation of HO-1. Furthermore, RGC death and levels of 4-hydroxy-2-nonenal (4HNE) were increased after optic nerve injury in vivo. Pretreatment with Nip significantly suppressed RGC death and accumulation of 4HNE after injury through an HO-1 activity-dependent mechanism. These data demonstrate a novel neuroprotective action of Nip against oxidative stress-induced RGC death in vitro and in vivo. © 2012 Elsevier Ltd. All rights reserved

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs\u27 program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders. © 2013 Koriyama et al

Neuritogenic activity of a genipin derivative in retinal ganglion cells is mediated by retinoic acid receptor β expression through nitric oxide/S-nitrosylation signaling

Genipin, a herbal iridoid, is known to have both neuroprotective and neuritogenic activity in neuronal cell lines. As it is structurally similar to tetrahydrobiopterin, its activity is believed to be nitric oxide (NO)-dependent. We previously proposed a novel neuroprotective activity of a genipin derivative, (1R)-isoPropyloxygenipin (IPRG001), whereby it reduces oxidative stress in RGC-5, a neuronal precursor cell line of retinal origin through protein S-nitrosylation. In the present study, we investigated another neuritogenic property of IPRG001 in RGC-5 cells and retinal explant culture where in we focused on the NO-cGMP-dependent and protein S-nitrosylation pathways. IPRG001 stimulated neurite outgrowth in RGC-5 cells and retinal explant culture through NO-dependent signaling, but not NO-dependent cGMP signaling. Neurite outgrowth with IPRG001 requires retinoic acid receptor β (RARβ) expression, which is suppressed by an RAR blocking agent and siRNA inhibition. Thereby, we hypothesized that RARβ expression is mediated by protein S-nitrosylation. S-nitrosylation of histone deacetylase 2 is a key mechanism in chromatin remodeling leading to transcriptional gene activation. We found a parallelism between S-nitrosylation of histone diacetylase 2 and the induction of RARβ expression with IPRG001 treatment. The both neuroprotective and neuritogenic activities of genipin could be a new target for the regeneration of retinal ganglion cells after glaucomatous conditions. © 2011 International Society for Neurochemistry

A novel function of neuroglobin for neuroregeneration in mice after optic nerve injury

金沢大学医薬保健研究域保健学系Neuroglobin (Ngb) is a recently discovered heme protein in the vertebrate brain that can bind to oxygen molecules. Mammalian Ngb plays a crucial role in neuroprotection under conditions of oxidative stress. To investigate other potential functions of Ngb, we investigated the mouse retinal Ngb system following optic nerve injury. In the retina of control mice, Ngb immunoreactivity was limited to the retinal ganglion cell (RGC) layer, and this immunoreactivity rapidly decreased to less than 50% of the control level 5 days after optic nerve injury. On the basis of this decrease, we designed in vivo experiments with enhanced expression of Ngb using adult mouse retina. The enhanced expression of Ngb was achieved by injecting chimeric human Ngb protein, which included the cell membrane-penetrating module of fish Ngb. One-day pretreatment with chimeric Ngb increased immunoreactivity levels of Ngb two-fold in mouse RGCs and increased the number of surviving RGCs three-fold by 14 days after optic nerve injury compared with vehicle controls. Furthermore, in the mouse retinas showing enhanced Ngb expression, several regenerating central optic axons exhibited outgrowth and were found to pass through the nerve crush site 14 days after nerve injury. No such regenerating optic axons were observed in the control mouse optic nerve during the same time frame. The data obtained from in vivo experiments strongly indicate that mammalian Ngb has neuroprotective and neuroregenerative properties. © 2017 Elsevier Inc.Embargo Period 12 month