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Article ; Online: Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5

Yosuke Hashimoto / Wei Zhou / Kohtaroh Hamauchi / Keisuke Shirakura / Takefumi Doi / Kiyohito Yagi / Tatsuya Sawasaki / Yoshiaki Okada / Masuo Kondoh / Hiroyuki Takeda

Scientific Reports, Vol 8, Iss 1, Pp 1-

2018 Volume 12

Abstract: Abstract The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly ... ...

Abstract	Abstract The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) and inhibited CLDN-5-containing tight junctions. These results demonstrate the effectiveness of the methods for monoclonal antibody development targeting difficult-to-produce membrane proteins such as CLDNs.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2018-05-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Structural Basis for PPARα Activation by 1H-pyrazolo-[3,4-b]pyridine Derivatives

Takuya Yoshida / Hiroya Oki / Michihiro Doi / Syohei Fukuda / Tomohiro Yuzuriha / Ryotaro Tabata / Kenji Ishimoto / Kazuki Kawahara / Tadayasu Ohkubo / Hiroyuki Miyachi / Takefumi Doi / Keisuke Tachibana

Scientific Reports, Vol 10, Iss 1, Pp 1-

2020 Volume 9

Abstract: Abstract Small-molecule agonism of peroxisome proliferator-activated receptor α (PPARα), a ligand-activated transcriptional factor involved in regulating fatty acid metabolism, is an important approach for treating dyslipidemia. Here, we determined the ... ...

Abstract	Abstract Small-molecule agonism of peroxisome proliferator-activated receptor α (PPARα), a ligand-activated transcriptional factor involved in regulating fatty acid metabolism, is an important approach for treating dyslipidemia. Here, we determined the structures of the ligand-binding domain (LBD) of PPARα in complex with 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives, which were recently identified as PPARα-selective activators with markedly different structures from those of the well-known PPARα agonists fibrates. The crystal structures of the complexes showed that they form a canonical hydrogen-bond network involving helix 12 in the LBD, which is thought to be essential for PPARα activation, as also observed for fibrates. However, the phenyl side chain of the compounds occupies a small cavity between Ile272 and Ile354, which is rarely accessed by fibrates. This unique feature may be essential for subtype selectivity and combine with the well-characterized binding mode of fibrates to improve activity. These findings demonstrate the advantage of using 1H-pyrazolo-[3,4-b]pyridine as a skeleton of PPARα agonists and provide insight into the design of molecules for treating dyslipidemia.
Keywords	Medicine ; R ; Science ; Q
Subject code	540
Language	English
Publishing date	2020-05-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The CalcR-PKA-Yap1 Axis Is Critical for Maintaining Quiescence in Muscle Stem Cells

Lidan Zhang / Yu-taro Noguchi / Hiroyuki Nakayama / Takayuki Kaji / Kazutake Tsujikawa / Madoka Ikemoto-Uezumi / Akiyoshi Uezumi / Yoshiaki Okada / Takefumi Doi / Shuichi Watanabe / Thomas Braun / Yasushi Fujio / So-ichiro Fukada

Cell Reports, Vol 29, Iss 8, Pp 2154-2163.e

2019 Volume 5

Abstract: Summary: Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor ( ... ...

Abstract	Summary: Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor (CalcR) is critical for sustaining quiescence in muscle satellite (stem) cells (MuSCs). However, the molecular mechanisms by which CalcR signaling regulates quiescence in MuSCs are enigmatic. Here, we demonstrate that transgenic expression of the catalytic domain of protein kinase A (PKA) restores the quiescence of CalcR-mutant MuSCs and delays MuSC activation. Mechanistically, CalcR-activated PKA phosphorylates Lats1/2, the main effector of Hippo signaling, thereby inhibiting the nuclear accumulation of Yap1, which prevents expression of Hippo-target genes, including cell-cycle-related molecules. Importantly, genetic inactivation of Yap1 in CalcR-mutant MuSCs reinstates quiescence in CalcR-mutant MuSCs, indicating that the CalcR-PKA-Lats1/2-Yap1 axis plays a critical role in sustaining MuSC quiescence. : Zhang et al. reveal the downstream pathway of CalcR in muscle stem cells. The CalcR-PKA axis phosphorylates Lats1/2, the main effector of Hippo signaling, and inhibits Yap1 nuclear accumulation. PKA activation or Yap1 inactivation restores CalcR-mutant muscle stem cell quiescence, indicating that the CalcR-PKA-Lats1/2-Yap1 axis is critical for sustaining MuSC quiescence. Keywords: Muscle stem cells, Calcitonin receptor, GPCR, PKA, Hippo, Quiescence, Yap
Keywords	Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2019-11-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The Role of PPARs in Cancer

Takefumi Doi / Kenji Ishimoto / Daisuke Yamasaki / Keisuke Tachibana

PPAR Research, Vol

2008 Volume 2008

Abstract: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARÃŽÂ± is mainly expressed in the liver, where it activates fatty acid catabolism. PPARÃŽÂ± ... ...

Abstract	Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARÃŽÂ± is mainly expressed in the liver, where it activates fatty acid catabolism. PPARÃŽÂ± activators have been used to treat dyslipidemia, causing a reduction in plasma triglyceride and elevation of high-density lipoprotein cholesterol. PPARÃŽÂ´ is expressed ubiquitously and is implicated in fatty acid oxidation and keratinocyte differentiation. PPARÃŽÂ´ activators have been proposed for the treatment of metabolic disease. PPARÃŽÂ³2 is expressed exclusively in adipose tissue and plays a pivotal role in adipocyte differentiation. PPARÃŽÂ³ is involved in glucose metabolism through the improvement of insulin sensitivity and represents a potential therapeutic target of type 2 diabetes. Thus PPARs are molecular targets for the development of drugs treating metabolic syndrome. However, PPARs also play a role in the regulation of cancer cell growth. Here, we review the function of PPARs in tumor growth.
Keywords	Internal medicine ; RC31-1245 ; Medicine ; R ; DOAJ:Internal medicine ; DOAJ:Medicine (General) ; DOAJ:Health Sciences ; Physiology ; QP1-981 ; Science ; Q ; DOAJ:Physiology ; DOAJ:Biology ; DOAJ:Biology and Life Sciences
Language	English
Publishing date	2008-06-01T00:00:00Z
Publisher	Hindawi Publishing Corporation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The Role of PPARs in Cancer

Keisuke Tachibana / Daisuke Yamasaki / Kenji Ishimoto / Takefumi Doi

PPAR Research, Vol

2008 Volume 2008

Abstract: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPAR𝛼 is mainly expressed in the liver, where it activates fatty acid catabolism. PPAR𝛼 activators ... ...

Abstract	Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPAR𝛼 is mainly expressed in the liver, where it activates fatty acid catabolism. PPAR𝛼 activators have been used to treat dyslipidemia, causing a reduction in plasma triglyceride and elevation of high-density lipoprotein cholesterol. PPAR𝛿 is expressed ubiquitously and is implicated in fatty acid oxidation and keratinocyte differentiation. PPAR𝛿 activators have been proposed for the treatment of metabolic disease. PPAR𝛾2 is expressed exclusively in adipose tissue and plays a pivotal role in adipocyte differentiation. PPAR𝛾 is involved in glucose metabolism through the improvement of insulin sensitivity and represents a potential therapeutic target of type 2 diabetes. Thus PPARs are molecular targets for the development of drugs treating metabolic syndrome. However, PPARs also play a role in the regulation of cancer cell growth. Here, we review the function of PPARs in tumor growth.
Keywords	Internal medicine ; RC31-1245 ; Medicine ; R ; Physiology ; QP1-981 ; Science ; Q ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2008-01-01T00:00:00Z
Publisher	Hindawi Publishing Corporation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Generation and characterization of a human–mouse chimeric antibody against the extracellular domain of claudin-1 for cancer therapy using a mouse model

Hashimoto, Yosuke / Minoru Tada / Manami Iida / Shotaro Nagase / Tomoyuki Hata / Akihiro Watari / Yoshiaki Okada / Takefumi Doi / Masayoshi Fukasawa / Kiyohito Yagi / Masuo Kondoh

Biochemical and biophysical research communications. 2016 Aug. 12, v. 477, no. 1

2016

Abstract: Claudin-1 (CLDN-1), an integral transmembrane protein, is an attractive target for drug absorption, prevention of infection, and cancer therapy. Previously, we generated mouse anti-CLDN-1 monoclonal antibodies (mAbs) and found that they enhanced ... ...

Abstract	Claudin-1 (CLDN-1), an integral transmembrane protein, is an attractive target for drug absorption, prevention of infection, and cancer therapy. Previously, we generated mouse anti-CLDN-1 monoclonal antibodies (mAbs) and found that they enhanced epidermal absorption of a drug and prevented hepatitis C virus infection in human hepatocytes. Here, we investigated anti-tumor activity of a human–mouse chimeric IgG1, xi-3A2, from one of the anti-CLDN-1 mAbs, clone 3A2. Xi-3A2 accumulated in the tumor tissues in mice bearing with human CLDN-1–expressing tumor cells. Xi-3A2 activated Fcγ receptor IIIa–expressing reporter cells in the presence of human CLDN-1–expressing cells, suggesting xi-3A2 has a potential to exhibit antibody-dependent cellular cytotoxicity against CLDN-1 expressing tumor cells. We also constructed a mutant xi-3A2 antibody with Gly, Ser, and Ile substituted with Ala, Asp, and Arg at positions 236, 239, and 332 of the Fc domain. This mutant antibody showed greater activation of Fcγ receptor IIIa and in vivo anti-tumor activity in mice bearing human CLDN-1-expressing tumors than xi-3A2 did. These findings indicate that the G236A/S239D/I332E mutant of xi-3A2 might be a promising lead for tumor therapy.
Keywords	Hepatitis C virus ; animal models ; antineoplastic activity ; chimerism ; cytotoxicity ; hepatocytes ; human diseases ; humans ; immunoglobulin G ; mice ; monoclonal antibodies ; mutants ; neoplasm cells ; neoplasms ; pharmacokinetics ; therapeutics ; transmembrane proteins
Language	English
Dates of publication	2016-0812
Size	p. 91-95.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2016.06.025
Database	NAL-Catalogue (AGRICOLA)

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Article: Endothelial Robo4 regulates IL-6 production by endothelial cells and monocytes via a crosstalk mechanism in inflammation

Shirakura, Keisuke / Miki Sakai / Nana Yamamoto / Naoya Shigesada / Nobumasa Hino / Ryosuke Ishiba / Shiori Manabe / Taito Kashio / Takefumi Doi / Toru Tanaka / William C. Aird / Yoshiaki Okada / Yu Fukushima

Biochemical and biophysical research communications. 2018 Jan. 01, v. 495, no. 1

2018

Abstract: Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in ... ...

Abstract	Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in inflammation remain unclear. To investigate physiological Robo4 functions in inflammation, we performed a loss-of-function study in vitro and in vivo using lipopolysaccharide (LPS)-induced endotoxemia models. Subcutaneous injection of LPS into Robo4-knockout mice reduced circulating IL-6 levels. siRNA-mediated Robo4 knockdown suppressed IL-6 production induced by LPS, IL-1β, and TNFα, in human umbilical vein endothelial cells (HUVECs). Coculture experiments with HUVECs and a monocytic cell line, U937 cells, demonstrated that Robo4 knockdown suppresses IL-6 production by both endothelial cells and U937 cells. Further coculture experiments demonstrated that Robo4 knockdown inhibited a novel IL-6 amplification mechanism mediated by crosstalk between endothelial cells and U937 cells via direct interactions and two mediators, GM-CSF and IL-1β. Taken together, we demonstrated novel Robo4 functions in inflammation, i.e., it promotes IL-6 production by endothelial cells and immune cells via crosstalk.
Keywords	angiogenesis ; cell lines ; coculture ; endotoxemia ; granulocyte-macrophage colony-stimulating factor ; human umbilical vein endothelial cells ; inflammation ; interleukin-1beta ; interleukin-6 ; lipopolysaccharides ; loss-of-function mutation ; mice ; models ; monocytes ; subcutaneous injection ; tumor necrosis factor-alpha
Language	English
Dates of publication	2018-0101
Size	p. 801-806.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2017.11.067
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Article: Facilitation of adipocyte differentiation of 3T3-L1 cells by debrominated tetrabromobisphenol A compounds detected in Japanese breast milk

Akiyama, Ema / Ayami Mizuno / Hideki Kakutani / Keisuke Tachibana / Osamu Aozasa / Ryota Sorakubo / Souichi Ohta / Takefumi Doi / Teruyuki Nakao / Yuka Motomura / Yuki Takano

Environmental Research. 2015 July, v. 140

2015

Abstract: Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2â²,6-tribromobisphenol A (TriBBPA), 2,2â²-dibromobisphenol A (2,2â²-DiBBPA), 2,6- ... ...

Abstract	Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2â²,6-tribromobisphenol A (TriBBPA), 2,2â²-dibromobisphenol A (2,2â²-DiBBPA), 2,6-dibromobisphenol A (2,6-DiBBPA), and 2-monobromobisphenol A (MoBBPA) and evaluated the actual extent of contamination with bisphenol A (BPA), TeBBPA and debrominated congeners in Japanese breast milk samples. TriBBPA was detected at higher levels than that of TeBBPA, while DiBBPA and MoBBPA were detected at lower levels than that of TeBBPA. This observation suggested that humans are exposed to debrominated congeners, which might cause adverse effects. Contamination of the congeners in breast milk was concern about risk infant health, having vulnerable defense system. As pilot study by in vitro experiment, we assessed the toxic potency of debrominated congeners by studying their effect on adipocyte differentiation in 3T3-L1 cells. We observed 2,6-DiBBPA, TriBBPA and TeBBPA elevated the lipid accumulation and adipocyte-specific protein 2 expression in a manner dependent on the number of substituted bromines. Moreover, PPARÎ³ transcriptional activities increased in a dose-dependent manner in the presence of 2,6-DiBBPA and TriBBPA as well as TeBBPA. Our study clarified that TeBBPA and its debrominated congeners accumulated in breast milk and the debrominated congeners promoted adipocyte differentiation, showing that a comprehensive evaluation of the influences of these compounds including the debrominated congeners of TeBBPA on health in infants is necessary.
Keywords	adipocytes ; adverse effects ; bisphenol A ; breast milk ; dose response ; flame retardants ; humans ; in vitro studies ; infants ; lipids ; risk ; toxicity ; transcription (genetics)
Language	English
Dates of publication	2015-07
Size	p. 157-164.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205699-9
ISSN	1096-0953 ; 0013-9351
ISSN (online)	1096-0953
ISSN	0013-9351
DOI	10.1016/j.envres.2015.03.035
Database	NAL-Catalogue (AGRICOLA)

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Article: Angubindin-1, a novel paracellular absorption enhancer acting at the tricellular tight junction

Krug, Susanne M / Akihiro Watari / Azusa Takahashi / Daisuke Iguchi / Hiroyuki Takeda / Kiyohito Yagi / Masahiro Nagahama / Masuo Kondoh / Michael Fromm / Takefumi Doi / Tatsuya Sawasaki / Tomohiro Hayaishi / Yoshiaki Okada

Journal of Controlled Release. 2017,

2017

Abstract: A limiting barrier for mucosal absorption of drugs is the tight junction (TJ). TJs exist between two adjacent cells (bicellular TJ, bTJ) and at the sites where three cells meet (tricellular TJ, tTJ). We present a novel approach which employs a ... ...

Abstract	A limiting barrier for mucosal absorption of drugs is the tight junction (TJ). TJs exist between two adjacent cells (bicellular TJ, bTJ) and at the sites where three cells meet (tricellular TJ, tTJ). We present a novel approach which employs a physiologically regulated pathway for the passage of large molecules through the tTJ. Main barrier-relevant tTJ proteins are tricellulin and angulin-1 to -3. We developed an angulin binder from Clostridium perfringens iota-toxin (Ib) whose receptor is angulin-1. An Ib fragment corresponding to amino acids 421â664 (Ib421-664) of iota-toxin proved to bind in cells expressing angulin-1 and -3, but not angulin-2. This binding led to removal of angulin-1 and tricellulin from the tTJ which enhanced the permeation of macromolecular solutes. Ib421-664 enhanced intestinal absorption in rats and mice. Our findings indicate that Ib421-664, which we designate angubindin-1, is a modulator of the tTJ barrier and that modulation of that barrier qualifies for a new strategy of developing a mucosal absorption enhancer.
Keywords	absorption ; amino acids ; chemical elements ; Clostridium perfringens ; drugs ; intestinal absorption ; mice ; proteins ; rats ; solutes ; tight junctions
Language	English
Size	p. .
Publishing place	Elsevier B.V.
Document type	Article
Note	Pre-press version
ZDB-ID	632533-6
ISSN	1873-4995 ; 0168-3659
ISSN (online)	1873-4995
ISSN	0168-3659
DOI	10.1016/j.jconrel.2017.05.024
Database	NAL-Catalogue (AGRICOLA)

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Article: Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase

Ishimoto, Kenji / Ayaka Hayase / Fumiko Kumagai / Hiroko Okuno / Juro Sakai / Keisuke Tachibana / Megumi Kawai / Nobumasa Hino / Takao Hamakubo / Takefumi Doi / Takeshi Kawamura / Tatsuhiko Kodama / Toshiya Tanaka / Yoshiaki Okada

Biochemical and biophysical research communications. 2017,

2017

Abstract: Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded ...

Abstract	Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded through its DSGXXS motif. We show that Lipin-1 interacts with either of two E3 ubiquitin ligases, BTRC or FBXW11, and that this interaction is DSGXXS-dependent and mediates the attachment of polyubiquitin chains. Further, we demonstrate that degradation of Lipin-1 is regulated by BTRC in the cytoplasm and on membranes. These novel insights into the regulation of human Lipin-1 stability will be useful in planning further studies to elucidate its metabolic processes.
Keywords	cytoplasm ; energy metabolism ; humans ; lipids ; planning ; ubiquitin-protein ligase
Language	English
Size	p. .
Publishing place	Elsevier Inc.
Document type	Article
Note	Pre-press version
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2017.04.159
Database	NAL-Catalogue (AGRICOLA)

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