研究者詳細 - 佐藤　あやの

論文 - 佐藤　あやの

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Uncovering a Latent Bioactive Interleukin-6 Glycoform. 査読国際誌

Yasuhiro Kajihara, Yanbo Liu, Yuta Maki, Ryo Okamoto, Ayano Satoh, Yasuto Todokoro, Yurie Kanemitsu, Keito Otani

Angewandte Chemie (International ed. in English) 63 ( 50 ) e202411213 2024年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

A bioinspired semisynthesis of human-interleukin-6 bearing N-glycan at Asn143 (143glycosyl-IL-6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide-backbone activation. 143Glycosyl-IL-6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6-polypeptide (1-141-position) with an intentionally inserted cysteine at 142-position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two-disulfide bonds (43-49 and 72-82). Utilizing the successfully formed free-142-cysteine, sequential conversions using cyanation of 142-cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1-141)-thioester. However, the resultant polypeptide-thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two-disulfide bonds of polypeptide (1-141)-thioester, an unprecedented hydrophilic N-glycan tag was inserted at the resultant cysteine thiols. The N-glycan tags greatly stabilized polypeptide-thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl-IL-6 polypeptide (183-amino acids). Removal of four N-glycan tags and immediate one-pot in vitro folding protocol efficiently produced the folded 143glycosyl-IL-6. The folded 143glycosyl-IL-6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl-IL-6.

DOI： 10.1002/anie.202411213

Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response 査読国際誌

Yuki M. Kyunai, Mika Sakamoto, Mayuko Koreishi, Yoshio Tsujino, Ayano Satoh

PLOS ONE 18 ( 2 ) e0281516 - e0281516 2023年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

GlycoMaple is a new tool to predict glycan structures based on the expression levels of 950 genes encoding glycan biosynthesis-related enzymes and proteins using RNA-seq data. The antioxidant response, protecting cells from oxidative stress, has been focused on because its activation may relieve pathological conditions, such as neurodegenerative diseases. Genes involved in the antioxidant response are defined within the GO:0006979 category, including 441 human genes. Fifteen genes overlap between the glycan biosynthesis-related genes defined by GlycoMaple and the antioxidant response genes defined by GO:0006979, one of which is FUT8. 5-Hydroxy-4-phenyl-butenolide (5H4PB) extracted from Chinese aromatic vinegar induces the expression of a series of antioxidant response genes that protect cells from oxidative stress via activation of the nuclear factor erythroid 2-related factor 2–antioxidant response element pathway. Here, we show that FUT8 is upregulated in both our RNA-seq data set of 5H4PB-treated cells and publicly available RNA-seq data set of cells treated with another antioxidant, sulforaphane. Applying our RNA-seq data set to GlycoMaple led to a prediction of an increase in the core fucose of N-glycan that was confirmed by flow cytometry using a fucose-binding lectin. These results suggest that FUT8 and core fucose expression may increase upon the antioxidant response.

DOI： 10.1371/journal.pone.0281516

A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi Apparatus After Experimental Manipulation 招待査読国際誌

Daisuke Takao, Yuki M. Kyunai, Yasushi Okada, Ayano Satoh

Methods in Molecular Biology 2557 275 - 285 2022年12月

担当区分：責任著者記述言語：英語掲載種別：論文集(書籍)内論文出版者・発行元：Springer US

The visual classification of cell images according to differences in the spatial patterns of subcellular structure is an important methodology in cell and developmental biology. Experimental perturbation of cell function can induce changes in the spatial distribution of organelles and their associated markers or labels. Here, we demonstrate how to achieve accurate, unbiased, high-throughput image classification using an artificial intelligence (AI) algorithm. We show that a convolutional neural network (CNN) algorithm can classify distinct patterns of Golgi images after drug or siRNA treatments, and we review our methods from cell preparation to image acquisition and CNN analysis.

DOI： 10.1007/978-1-0716-2639-9_18

An Electron Tomographic Analysis of Giantin-Deficient Golgi Proposes a New Function of the Golgin Protein Family 招待査読国際誌

Ayano Satoh, Mitsuko Hayashi-Nishino, Kunihiko Nishino

Methods in Molecular Biology 2557 235 - 246 2022年12月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：論文集(書籍)内論文出版者・発行元：Springer US

The Golgi apparatus is an organelle that mediates modifications, sorting, and transport of proteins and lipids. Golgins are a group of proteins with coiled-coil structures that localize to the Golgi and are thought to function as tethers to facilitate the docking of vesicles, Rab GTPases, and cytoskeleton components to the Golgi stack. Giantin is the longest golgin and has been thought to function as a tether for COPI vesicles along with other golgins, such as p115 and GM130. Contrary to our expectation that the loss of the tether will result in an increase in untethered COPI vesicles in the cytoplasm, our electron microscopy observations showed that the fenestrae normally present in Golgi cisternae were reduced upon Giantin knockdown. We also found that this structural change is accompanied by altered secretion of cargo proteins and cell surface glycosylation. These results indicate that there exists a correlation between Golgi structural changes caused by the loss of Giantin and Golgi function. Here, we describe electron tomography methods for the detection of structural changes in the Golgi.

DOI： 10.1007/978-1-0716-2639-9_15

Evaluation of skin sensitization based on interleukin‑2 promoter activation in Jurkat cells 査読国際誌

Taichi Nagahata, Yoshio Tsujino, Eiji Takayama, Haruka Hikasa, Ayano Satoh

Biomedical Reports 16 ( 1 ) 3 - 3 2021年11月

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

Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T-cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T-cell activation to supplement data on skin sensitization related to KE4. To evaluate T-cell activation, the Jurkat T-cell line stably expressing luciferase downstream of the pro-inflammatory cytokine interleukin-2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T-cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno-suppressiveness of chemical substances and supplements KE4 T-cell activation data, and may thus contribute to reducing the use of animal experiments.

DOI： 10.3892/br.2021.1486

Glycoprotein Semisynthesis by Chemical Insertion of Glycosyl Asparagine Using a Bifunctional Thioacid-Mediated Strategy 査読国際誌

Kota Nomura, Yuta Maki, Ryo Okamoto, Ayano Satoh, Yasuhiro Kajihara

Journal of the American Chemical Society 143 ( 27 ) 10157 - 10167 2021年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Chemical Society (ACS)

Glycosylation is a major modification of secreted and cell surface proteins, and the resultant glycans show considerable heterogeneity in their structures. To understand the biological processes arising from each glycoform, the preparation of homogeneous glycoproteins is essential for extensive biological experiments. To establish a more robust and rapid synthetic route for the synthesis of homogeneous glycoproteins, we studied several key reactions based on amino thioacids. We found that diacyl disulfide coupling (DDC) formed with glycosyl asparagine thioacid and peptide thioacid yielded glycopeptides. This efficient coupling reaction enabled us to develop a new glycoprotein synthesis method, such as the bifunctional thioacid-mediated strategy, which can couple two peptides with the N- and C-termini of glycosyl asparagine thioacid. Previous glycoprotein synthesis methods required valuable glycosyl asparagine in the early stage and subsequent multiple glycoprotein synthesis routes, whereas the developed concept can generate glycoproteins within a few steps from peptide and glycosyl asparagine thioacid. Herein, we report the characterization of the DDC of amino thioacids and the efficient ability of glycosyl asparagine thioacid to be used for robust glycoprotein semisynthesis.

DOI： 10.1021/jacs.1c02601

Synthesis and characterization of conductive flexible cellulose carbon nanohorn sheets for human tissue applications 査読国際誌

Karthik Paneer Selvam, Taichi Nagahata, Kosuke Kato, Mayuko Koreishi, Toshiyuki Nakamura, Yoshimasa Nakamura, Takeshi Nishikawa, Ayano Satoh, Yasuhiko Hayashi

Biomaterials Research 24 ( 1 ) 18 - 18 2020年10月

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

Abstract

Background

Conductive sheets of cellulose and carbon nanomaterials and its human skin applications are an interesting research aspect as they have potential for applications for skin compatibility. Hence it is needed to explore the effects and shed light on these applications.

Method

To fabricate wearable, portable, flexible, lightweight, inexpensive, and biocompatible composite materials, carbon nanohorns (CNHs) and hydroxyethylcellulose (HEC) were used as precursors to prepare CNH-HEC (Cnh-cel) composite sheets. Cnh-cel sheets were prepared with different loading concentrations of CNHs (10, 20 50,100 mg) in 200 mg cellulose. To fabricate the bio-compatible sheets, a pristine composite of CNHs and HEC was prepared without any pretreatment of the materials.

Results

The obtained sheets possess a conductivity of 1.83 × 10^− 10 S/m and bio-compatible with human skin. Analysis for skin-compatibility was performed for Cnh-cel sheets by h-CLAT in vitro skin sensitization tests to evaluate the activation of THP-1 cells. It was found that THP-1 cells were not activated by Cnh-cel; hence Cnh-cel is a safe biomaterial for human skin. It was also found that the composite allowed only a maximum loading of 100 mg to retain the consistent geometry of free-standing sheets of < 100 μm thickness. Since CNHs have a unique arrangement of aggregates (dahlia structure), the composite is homogeneous, as verified by transmission electron microscopy (TEM) and, scanning electron microscopy (SEM), and other functional properties investigated by Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), conductivity measurement, tensile strength measurement, and skin sensitization.

Conclusion

It can be concluded that cellulose and CNHs sheets are conductive and compatible to human skin applications.

DOI： 10.1186/s40824-020-00194-3

その他リンク： http://link.springer.com/article/10.1186/s40824-020-00194-3/fulltext.html

High-throughput screening of bioactive compounds via new catalytic reaction in the pooled mixture. 査読国際誌

Ayano Satoh, Yuta Nishina

Bioorganic & medicinal chemistry letters 29 ( 19 ) 126539 - 126539 2019年10月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

To increase the chances of finding new candidate molecules with medicinal properties, while expending less resource and effort, the present study used pooled substrates as starting materials. A bisindole compound that showed inhibitory activity was then isolated from the mixture, and the activity was improved by optimizing the substituents on the indole skeleton.

DOI： 10.1016/j.bmcl.2019.06.061

The Golgin Protein Giantin Regulates Interconnections Between Golgi Stacks 招待査読国際誌

Ayano Satoh, Mitsuko Hayashi-Nishino, Takuto Shakuno, Junko Masuda, Mayuko Koreishi, Runa Murakami, Yoshimasa Nakamura, Toshiyuki Nakamura, Naomi Abe-Kanoh, Yasuko Honjo, Joerg Malsam, Sidney Yu, Kunihiko Nishino

Frontiers in Cell and Developmental Biology 7 160 - 160 2019年8月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers Media SA

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi.

DOI： 10.3389/fcell.2019.00160

A RasGAP, DAB2IP, regulates lipid droplet homeostasis by serving as GAP toward RAB40C 査読国際誌

Xiaomin Luo, Chunman Li, Ran Tan, Xiaohui Xu, William K.K. Wu, Ayano Satoh, Tuanlao Wang, Sidney Yu

Oncotarget 8 ( 49 ) 85415 - 85427 2017年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Impact Journals, LLC

Lipid droplet (LD) homeostasis involves activities of various RAB small GTPases. Recently, we found RAB40C was one of the RAB proteins regulating LD homeostasis. RAB40C contains a unique SOCS domain that is required for clustering of LDs. However, its precise functional role in LD homeostasis and mechanism of regulation remain largely unknown. In this study, we observed over-accumulation of LDs in cells with RAB40C deleted by Crispr-Cas9 editing. RAB40C appeared to reduce LD accumulation after long term incubation of cells with oleic acid (24 hours). Unexpectedly, we found that Ras GTPase activating protein (GAP), DAB2IP, bound to RAB40C mainly via its GAP domain and could serve as RAB40C GAP. Studies involving overexpression of DAB2IP and its GAP defective mutant and siRNA depletion of DAB2IP all confirmed that DAB2IP negatively regulated the effect of RAB40C on LD homeostasis. These results provide a novel perspective on the regulation of RAB40C and implicate various signalling pathways regulated by DAB2IP, which may play a role in LD homeostasis via RAB40C.

DOI： 10.18632/oncotarget.19960

ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic 査読国際誌

Wenjia Gan, Caiyun Zhang, Ka Yu Siu, Ayano Satoh, Julian A. Tanner, Sidney Yu

BMC Cell Biology 18 ( 1 ) 22 - 22 2017年5月

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

BACKGROUND: Autophagy is an inducible autodigestive process that allows cells to recycle proteins and other materials for survival during stress and nutrient deprived conditions. The kinase ULK1 is required to activate this process. ULK1 phosphorylates a number of target proteins and regulates many cellular processes including the early secretory pathway. Recently, ULK1 has been demonstrated to phosphorylate Sec16 and affects the transport of serotonin transporter at the ER exit sites (ERES), but whether ULK1 may affect the transport of other cargo proteins and general secretion has not been fully addressed. RESULTS: In this study, we identified Sec23A, a component of the COPII vesicle coat, as a target of ULK1 phosphorylation. Elevated autophagy, induced by amino acid starvation, rapamycin, or overexpression of ULK1 caused aggregation of the ERES, a region of the ER dedicated for the budding of COPII vesicles. Transport of cargo proteins was also inhibited under these conditions and was retained at the ERES. ULK1 phosphorylation of Sec23A reduced the interaction between Sec23A and Sec31A. We identified serine 207, serine 312 and threonine 405 on Sec23A as ULK1 phosphorylation sites. Among these residues, serine 207, when changed to phospho-deficient and phospho-mimicking mutants, most faithfully recapitulated the above-mentioned effects of ULK1 phospho-regulation. CONCLUSION: These findings identify Sec23A as a new target of ULK1 and uncover a mechanism of coordinating intracellular protein transport and autophagy.

DOI： 10.1186/s12860-017-0138-8

An endoplasmic reticulum protein, Nogo‐B, facilitates alcoholic liver disease through regulation of kupffer cell polarization 査読国際誌

Jin‐Kyu Park, Mingjie Shao, Moon Young Kim, Soon Koo Baik, Mee Yon Cho, Teruo Utsumi, Ayano Satoh, Xinsho Ouyang, Chuhan Chung, Yasuko Iwakiri

Hepatology 65 ( 5 ) 1720 - 1734 2017年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Ovid Technologies (Wolters Kluwer Health)

Nogo‐B (Reticulon 4B) is an endoplasmic reticulum (ER) resident protein that regulates ER structure and function. Because ER stress is known to induce M2 macrophage polarization, we examined whether Nogo‐B regulates M1/M2 polarization of Kupffer cells and alters the pathogenesis of alcoholic liver disease (ALD). M1 and M2 phenotypes were assessed in relation to Nogo‐B expression and disease severity in liver specimens from ALD patients (NCT01875211). Liver specimens from wild‐type (WT) and Nogo‐B knockout (KO) mice fed a control or Lieber‐DeCarli ethanol liquid diet (5% ethanol) for 6 weeks were analyzed for liver injury and steatosis. Kupffer cells isolated from WT and Nogo‐B KO mice were assessed for M1 and M2 activation. A significant positive correlation was observed between Nogo‐B positive Kupffer cells and disease severity in ALD patients (n = 30, r = 0.66, P = 0.048). Furthermore, Nogo‐B–positive Kupffer cells were correlated with M1 activation (inducible nitric oxide synthase) (r = 0.50, P = 0.05) and negatively with markers of M2 status (CD163) (r = −0.48, P = 0.07) in these patients. WT mice exhibited significantly increased liver injury (P < 0.05) and higher hepatic triglyceride levels (P < 0.01) compared with Nogo‐B KO mice in response to chronic ethanol feeding. Nogo‐B in Kupffer cells promoted M1 polarization, whereas absence of Nogo‐B increased ER stress and M2 polarization in Kupffer cells. Conclusion: Nogo‐B is permissive of M1 polarization of Kupffer cells, thereby accentuating liver injury in ALD in humans and mice. Nogo‐B in Kupffer cells may represent a new therapeutic target for ALD. (Hepatology 2017;65:1720‐1734).

DOI： 10.1002/hep.29051

<scp>COPI</scp>–<scp>TRAPPII</scp> activates Rab18 and regulates its lipid droplet association 査読国際誌

Chunman Li, Xiaomin Luo, Shan Zhao, Gavin KY Siu, Yongheng Liang, Hsiao Chang Chan, Ayano Satoh, Sidney SB Yu

The EMBO Journal 36 ( 4 ) 441 - 457 2016年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：EMBO

Abstract

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

DOI： 10.15252/embj.201694866

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.15252/embj.201694866

Cellular distribution of injected PLGA-nanoparticles in the liver 査読国際誌

Jin-Kyu Park, Teruo Utsumi, Young-Eun Seo, Yang Deng, Ayano Satoh, William Mark Saltzman, Yasuko Iwakiri

Nanomedicine: Nanotechnology, Biology and Medicine 12 ( 5 ) 1365 - 1374 2016年7月

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

The cellular fate of nanoparticles in the liver is not fully understood. Because the effectiveness and safety of nanoparticles in liver therapy depends on targeting nanoparticles to the right cell populations, this study aimed to determine a relative distribution of PLGA-nanoparticles (sizes 271±1.4 nm) among liver cells in vivo. We found that Kupffer cells were the major cells that took up nanoparticles, followed by liver sinusoidal endothelial cells and hepatic stellate cells. Nanoparticles were found in only 7% of hepatocytes. Depletion of Kupffer cells by clodronate liposomes increased nanoparticle retention in liver sinusoidal endothelial cells and hepatic stellate cells, but not in hepatocytes. It is importantly suggested that studies of drug-loaded nanoparticle delivery to the liver have to demonstrate not only uptake of nanoparticles by the target cell type but also non-uptake by other cell types to assess their effect as well as ensure their safety.

DOI： 10.1016/j.nano.2016.01.013

Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation 査読国際誌

Kimie Date, Ayano Satoh, Kaoruko Iida, Haruko Ogawa

Journal of Biological Chemistry 290 ( 28 ) 17439 - 17450 2015年7月

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

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na(+)/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104-23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption stage in the intestine.

DOI： 10.1074/jbc.m114.594937

The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus 査読国際誌

Mayuko Koreishi, Thomas J. Gniadek, Sidney Yu, Junko Masuda, Yasuko Honjo, Ayano Satoh

PLoS ONE 8 ( 3 ) e59821 - e59821 2013年3月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.

DOI： 10.1371/journal.pone.0059821

Stabilization of Actin Bundles by a Dynamin 1/Cortactin Ring Complex Is Necessary for Growth Cone Filopodia 査読国際誌

Hiroshi Yamada, Tadashi Abe, Ayano Satoh, Nana Okazaki, Shota Tago, Kinue Kobayashi, Yumi Yoshida, Yoshiya Oda, Masami Watanabe, Kazuhito Tomizawa, Hideki Matsui, Kohji Takei

The Journal of Neuroscience 33 ( 10 ) 4514 - 4526 2013年3月

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

Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1–cortactin complexes appeared as “open” or “closed” rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol.In vitroincubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1–cortactin complex.

DOI： 10.1523/jneurosci.2762-12.2013

Absence of Nogo-B (Reticulon 4B) Facilitates Hepatic Stellate Cell Apoptosis and Diminishes Hepatic Fibrosis in Mice 査読国際誌

Keitaro Tashiro, Ayano Satoh, Teruo Utsumi, Chuhan Chung, Yasuko Iwakiri

The American Journal of Pathology 182 ( 3 ) 786 - 795 2013年3月

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

Nogo-B (reticulon 4B) accentuates hepatic fibrosis and cirrhosis, but the mechanism remains unclear. The aim of this study was to identify the role of Nogo-B in hepatic stellate cell (HSC) apoptosis in cirrhotic livers. Cirrhosis was generated by carbon tetrachloride inhalation in wild-type (WT) and Nogo-A/B knockout (Nogo-B KO) mice. HSCs were isolated from WT and Nogo-B KO mice and cultured for activation and transformation to myofibroblasts (MF-HSCs). Human hepatic stellate cells (LX2 cells) were used to assess apoptotic responses of activated HSCs after silencing or overexpressing Nogo-B. Livers from cirrhotic Nogo-B KO mice showed significantly reduced fibrosis (P < 0.05) compared with WT mice. Apoptotic cells were more prominent in fibrotic areas of cirrhotic Nogo-B KO livers. Nogo-B KO MF-HSCs showed significantly increased levels of apoptotic markers, cleaved poly (ADP-ribose) polymerase, and caspase-3 and -8 (P < 0.05) compared with WT MF-HSCs in response to staurosporine. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, increased cleaved caspase-3 and -8 levels in Nogo-B KO MF-HSCs compared with WT MF-HSCs (P < 0.01). In LX2 cells, Nogo-B knockdown enhanced apoptosis in response to staurosporine, whereas Nogo-B overexpression inhibited apoptosis. The absence of Nogo-B enhances apoptosis of HSCs in experimental cirrhosis. Selective blockade of Nogo-B in HSCs may represent a potential therapeutic strategy to mitigate liver fibrosis.

DOI： 10.1016/j.ajpath.2012.11.032

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking 国際誌

Mayuko Koreishi, Sidney Yu, Mayumi Oda, Yasuko Honjo, Ayano Satoh

PLoS ONE 8 ( 1 ) e54382 - e54382 2013年1月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking.

DOI： 10.1371/journal.pone.0054382

TRAPPC9 Mediates the Interaction between p150Glued and COPII Vesicles at the Target Membrane 査読国際誌

Min Zong, Ayano Satoh, Mei Kuen Yu, Ka Yu Siu, Wing Yan Ng, Hsiao Chang Chan, Julian A. Tanner, Sidney Yu

PLoS ONE 7 ( 1 ) e29995 - e29995 2012年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

BACKGROUND: The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150(Glued), a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown. PRINCIPLE FINDINGS: We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150(Glued) away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150(Glued) via the same carboxyl terminal domain of p150(Glued) that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150(Glued) and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150(Glued) from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane. CONCLUSIONS: These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.

DOI： 10.1371/journal.pone.0029995

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