Language：English   Publisher：Journal of the American Chemical Society  

Site-selective cleavage of the peptide backbone in proteins is an important class of post-translational modification (PTM) in nature. However, the organic chemistry for such site-selective peptide bond cleavages has yet to be fully explored. Herein, we report cysteine S-formylation as a means of selective protein backbone cleavage. We developed N-formyl sulfonylanilide as a cysteine-selective formylation reagent for peptides and proteins. Upon S-formylation with the reagent, the amide bond adjacent to the S-formylated cysteine is cleaved by hydrolysis under neutral aqueous conditions. Formylation probes bearing a protein ligand enabled the affinity-based selective cleavage of the target proteins not only in the test tube but also under biorelevant conditions such as in crude cell lysate and on the cell surface. These results demonstrate the high biocompatibility of this protein cleavage technology. A proof-of-concept study of cleavage-induced protein activation further demonstrates its utility as a platform for the functional regulation of proteins by artificial PTM.

DOI： 10.1021/jacs.4c10991

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DOI： 10.1093/jb/mvae092

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

DOI： 10.1016/j.bbrc.2024.150077

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Journal of Physiology - Renal Physiology  

Increased dietary phosphate consumption intensifies renal phosphate burden. Several mechanisms for phosphate-induced renal tubulointerstitial fibrosis have been reported. Considering the dual nature of phosphate as both a potential renal toxin and an essential nutrient for the body, kidneys may possess inherent protective mechanisms against phosphate overload, rather than succumbing solely to injury. However, there is limited understanding of such mechanisms. To identify these mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) analysis of the kidneys of control and dietary phosphate-loaded (Phos) mice at a time point when the Phos group had not yet developed tubulointerstitial fibrosis. scRNA-seq analysis identified the highest number of differentially expressed genes in the clusters belonging to proximal tubular epithelial cells (PTECs). Based on these differentially expressed genes, in silico analyses suggested that the Phos group activated peroxisome proliferator-activated receptor-α (PPAR-α) and fatty acid β-oxidation (FAO) in the PTECs. This activation was further substantiated through various experiments, including the use of an FAO activity visualization probe. Compared with wild-type mice, Ppara knockout mice exhibited exacerbated tubulointerstitial fibrosis in response to phosphate overload. Experiments conducted with cultured PTECs demonstrated that activation of the PPAR-α/FAO pathway leads to improved cellular viability under high-phosphate conditions. The Phos group mice showed a decreased serum concentration of free fatty acids, which are endogenous PPAR-α agonists. Instead, experiments using cultured PTECs revealed that phosphate directly activates the PPAR-α/FAO pathway. These findings indicate that noncanonical metabolic reprogramming via endogenous activation of the PPAR-α/FAO pathway in PTECs is essential to counteract phosphate toxicity.NEW & NOTEWORTHY This study revealed the activation of peroxisome proliferator-activated receptor-α and fatty acid β-oxidation in proximal tubular epithelial cells as an endogenous mechanism to protect the kidney from phosphate toxicity. These findings highlight noncanonical metabolic reprogramming as a potential target for suppressing phosphate toxicity in the kidneys.

DOI： 10.1152/ajprenal.00046.2024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Fluorescent probes are indispensable tools for detection of enzymatic activities. However, detection of enzymes with high substrate specificity with substrate-based fluorescent probes is still challenging, due to their relatively large molecular size. We report herein enzyme assays coupled with a bioorthogonal fluoride ion-induced desilylation of fluorogenic probe. This sensing system is useful for in situ detection of enzyme reactions with high substrate specificity.

DOI： 10.1093/chemle/upae031

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Repository Public URL： https://hdl.handle.net/2324/7337634

Language：English   Publisher：(公社)日本薬学会  

加齢黄斑変性患者の網膜に蓄積するドルーゼンの主要成分である酸化LDL(oxLDL)に着目し、oxLDLが網膜色素上皮(RPE)細胞の代謝に及ぼす影響について検討した。ox-LDLへの長時間曝露はRPE細胞の脂肪酸β酸化(FAO)、酸化的リン酸化および解糖活性を変化させ、ミトコンドリアの活性酸素種産生を増加させた。代謝に対する影響は、ox-LDL処理の濃度および時間により変化した。多能性幹細胞由来RPE細胞と比べてARPE-19細胞のバリア機能およびFAO活性は低く、網膜疾患の研究にARPE-19細胞を用いることの限界が示された。

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Pharmaceutical Society of Japan  

<p>Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid β-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.</p>

DOI： 10.1248/bpb.b23-00849

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Journal of Physiology - Endocrinology and Metabolism  

Mitochondrial fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes β-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(N-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution.NEW & NOTEWORTHY Fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.

DOI： 10.1152/ajpendo.00147.2023

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1152/ajpendo.00147.2023

Language：English   Publishing type：Research paper (scientific journal)  

脂肪酸β酸化の活性を培養細胞やマウス肝臓で検出可能な初めてのケミカルプローブの開発に成功した。

DOI： doi.org/10.1021/jacs.3c02043

Language：English   Publishing type：Research paper (scientific journal)  

生体内において金属イオンを選択的に検出する蛍光センサーの開発は、金属イオンの生理学的意義を評価するために重要である。我々はこれまでarene-metal ion contact　（AMコンタクト）と命名した新しい金属イオン検出システムを考案したが、検出できる金属イオンが限られていた。今回我々はAMコンタクトで検出可能な金属イオンを拡張するための新しい分子デザインを考案し、生細胞表層や生細胞内での様々な金属イオンの検出に成功した。特に今回の設計では標的イオンをレシオ検出可能な分子設計であり、定量性の高い検出が可能であるという特徴がある。さらに、今回の設計では利用可能な蛍光色素（蛍光波長）の拡張にも成功しており、細胞内での金属イオンの検出に大きく貢献できると考えられる。

DOI： 10.1038/s42004-021-00541-y

Language：Japanese   Publishing type：Research paper (scientific journal)  

蛍光プローブは様々な酵素活性をリアルタイムかつ高感度に検出可能であることから、これまで盛んに研究されている。しかし、検出可能な酵素は、検出メカニズムが確立されている類似の酵素群に限られているのが現状である。そのため新しい検出メカニズムの開発が常に求められている。本研究では酵素反応によってカルボン酸を生成する酵素を検出するための新しい検出メカニズムを開発した。本システムでは、トリメチル化カルバメート基を骨格とする新しいセルフイモレーティブリンカーを開発し、酵素反応によるカルボン酸の生成とそれに続く分子内環化反応によって蛍光色素が脱離し蛍光onとなる。本システムを用いて、細胞内での加水分解酵素などの検出に成功した。

Language：Japanese   Publishing type：Research paper (scientific journal)  

電子顕微鏡はタンパク質を1分子レベルでイメージング可能な高い分解能を有する、有用なイメージング法である。電子顕微鏡で標的タンパク質を検出するためには金ナノ粒子などを標識する必要があるが、従来の抗体を用いた標識法では抗体のサイズが大きいことが原因で、標的タンパク質と金ナノ粒子が離れてしまう。そのため、標的タンパク質の局在を正確にイメージングできないという大きな問題があった。今回我々は、これまで我々が開発したペプチドタグによるタンパク質特異的ラベル化法を、電子顕微鏡イメージングに応用した。本手法では抗体よりはるかにサイズの小さなペプチドを用いるため、従来法の問題を解決することが可能である。実際我々は、細胞表層に発現させたペプチドタグ導入タンパク質に金ナノ粒子を特異的に標識し、本手法が抗体修飾法と比較して優れていることを示した。

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1246/bcsj.20190034

Language：English   Publishing type：Research paper (scientific journal)  

細胞表層に存在する様々な受容体は、シグナル伝達をはじめ様々な生命現象に深く関与する。受容体が機能を発現する際は受容体の局在が大きく変化するため、その機能解析のためには局在変化を追跡することが必要である。しかし、これまでの細胞を固定化し抗体で受容体を標識する免疫染色法では、生細胞での挙動をリアルタイムに解析することが困難であった。今回我々は、生細胞で内在性受容体を特異的に蛍光標識し、その挙動をリアルタイムに解析可能な新しいケミカルツールを開発した。

Language：English   Publishing type：Research paper (scientific journal)  

Hydropersulfide (R–SSH) is an important class of reactive sulfur species (RSS) involved in a variety of physiological processes in mammals. A fluorescent probe capable of real-time detection of hydropersulfide levels in living cells would be a versatile tool to elucidate its roles in cell signalling and redox homeostasis. In this paper, we report a ratiometric fluorescent probe for hydropersulfide sensing, based on a fluorescence resonance energy transfer (FRET) mechanism. This sensing mechanism involves a nucleophilic reaction of a hydropersulfide with the pyronine-unit of the probe, which modulates the intramolecular FRET efficiency to induce a dual-emission change. The reversible nature of this reaction allows us to detect increases and decreases of hydropersulfide levels in a real-time manner. The probe fluorometrically sensed highly reactive hydropersulfides, such as H2S2 and Cys-SSH, while the fluorescence response to biologically abundant cysteine and glutathione was negligible. Taking advantage of the reversible and selective sensing properties, this probe was successfully applied to the ratiometric imaging of concentration dynamics of endogenously produced hydropersulfides in living cells.

DOI： 10.1039/c6sc03856e

Language：English   Publishing type：Research paper (scientific journal)  

Reported is a new fluorescent probe based on boron–dipyrromethene dye (BODIPY) for late endosome staining in live cells. Among the 14 pH-responsive BODIPY-based far-red/near-infrared dyes used, it was found that a (4-methyl-1-piperazinyl)phenyl-substituted BODIPY could stain late endosomes in three different cell lines. This BODIPY dye was applicable for measurement of the fusion time of late endosomes and lysosomes, thereby demonstrating the utility of this dye for functional analyses of late endosomes in live cells.

DOI： 10.1002/cplu.201600249

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c6cc03336a

Language：English   Publishing type：Research paper (scientific journal)  

Control of fluorescent dye localization in live cells is crucial for fluorescence imaging. Here, we describe quantitative structure activity relation (QSAR) models for predicting intracellular localization of fluorescent dyes. For generating the QSAR models, electric charge (Z) calculated by pKa, conjugated bond number (CBN), the largest conjugated fragment (LCF), molecular weight (MW) and log P were used as parameters. We identified the intracellular localization of 119 BODIPY dyes in live NIH3T3 cells, and assessed the accuracy of our models by comparing their predictions with the observed dye localizations. As predicted by the models, no BODIPY dyes localized in nuclei or plasma membranes. The accuracy of the model for localization in fat droplets was 92%, with the models for cytosol and lysosomes showing poorer agreement with observed dye localization, albeit well above chance levels. Overall therefore the utility of QSAR models for predicting dye localization in live cells was clearly demonstrated.

DOI： 10.2174/1386207319666160408150528

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c1cc16263b

Language：English   Publishing type：Research paper (scientific journal)  

Event date： 2022.3

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Venue：オンライン   Country：Japan  

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Venue：東京大学弥生キャンパス   Country：Japan  

Event date： 2019.9

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Venue：東北大学青葉山東キャンパス   Country：Japan  

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Venue：ウインクあいち   Country：Japan  

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Venue：東京医科歯科大学M&Dタワー　2階　鈴木章夫記念講堂   Country：Japan  

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Venue：日本大学理工学部　船橋キャンパス   Country：Japan  

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Venue：日本大学理工学部　船橋キャンパス   Country：Japan  

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Venue：日本大学理工学部　船橋キャンパス   Country：Japan  

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Venue：日本大学理工学部　船橋キャンパ   Country：Japan  

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Venue：崇城大学   Country：Japan  

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Venue：東京大学　弥生キャンパス   Country：Japan  

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Venue：慶應義塾大学　日吉キャンパス   Country：Japan  

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Venue：慶應義塾大学　日吉キャンパス   Country：Japan  

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Venue：同志社大学　京田辺キャンパス   Country：Japan  

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Venue：同志社大学　京田辺キャンパス   Country：Japan  

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Venue：同志社大学　京田辺キャンパス   Country：Japan  

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Type：Competition, symposium, etc. 

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Type：Competition, symposium, etc. 

日本化学会生体機能関連化学部会　若手幹事

日本化学会生体機能関連化学部会　若手幹事

日本化学会生体機能関連化学部会　若手幹事