研究者詳細 - 木下　理恵

Identification of an S100A8 Receptor Neuroplastin-β and its Heterodimer Formation with EMMPRIN 査読

Masakiyo Sakaguchi, Mami Yamamoto, Masashi Miyai, Tatsuo Maeda, Junichiro Hiruma, Hitoshi Murata, Rie Kinoshita, I. Made, Winarsa Ruma, ndy Widya Putranto, Yusuke Inoue, Shin Morizane, Nam Ho Huh, Ryoji Tsuboi, Toshihiko Hibino

Journal of Investigative Dermatology, Advances in biology of skin 136 ( 11 ) 2240 - 2250 2016年11月

MCAM, as a novel receptor for S100A8/A9, mediates progression of malignant melanoma through prominent activation of NF-κB and ROS formation upon ligand binding 査読

I. Made, Winarsa Ruma, ndy Widya Putranto, Eisaku Kondo, Hitoshi Murata, Masami Watanabe, Peng Huang, Rie Kinoshita, Junichiro Futami, Yusuke Inoue, Akira Yamauchi, I. Wayan, Sumardika,Chen Youyi, Ken Ichi Yamamoto, Yasutomo Nasu, Masahiro Nishibori, Toshihiko H

Clinical and Experimental Metastasis 33 ( 6 ) 609 - 627 2016年8月

An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli 査読

Junichiro Futami, Yuki Atago, Akari Azuma, Endy Widya Putranto, Rie Kinoshita, Hitoshi Murata, Masakiyo Sakaguchi

Biochemistry and Biophysics Reports 6 94 - 100 2016年7月

記述言語：英語出版者・発行元：Elsevier B.V.

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.

DOI： 10.1016/j.bbrep.2016.03.009

Scopus

S-カチオン化全長・水溶性抗原を用いた高感度抗体技術による腫瘍免疫応答の定量評価

二見淳一郎, 野々村英典, 木戸桃子, 新土居奈緒美, 藤枝奈緒, 細井亮宏, 藤田佳那, 万袋木麻子, 愛宕祐基, 木下理恵, 本荘知子, 松下博一, 上中明子, 中山睿一, 垣見和宏

日本生物工学会大会講演要旨集平成27年度 191 - 191 2015年9月

The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression 査読国際誌

Rie Kinoshita, Masami Watanabe, Peng Huang, Shun-Ai Li, Masakiyo Sakaguchi, Hiromi Kumon, Junichiro Futami

ONCOLOGY REPORTS 33 ( 6 ) 2908 - 2914 2015年6月

担当区分：筆頭著者記述言語：英語出版者・発行元：SPANDIDOS PUBL LTD

Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3 beta (GSK-3 beta) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

DOI： 10.3892/or.2015.3885

A vaccine strategy with multiple prostatic acid phosphatase-fused cytokines for prostate cancer treatment 査読国際誌

Kei Fujio, Masami Watanabe, Hideo Ueki, Shun-Ai Li, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Toyohiko Watanabe, Yasutomo Nasu, Hiromi Kumon

ONCOLOGY REPORTS 33 ( 4 ) 1585 - 1592 2015年4月

記述言語：英語出版者・発行元：SPANDIDOS PUBL LTD

Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic antigen-vaccination strategy to enhance the therapeutic effects.

DOI： 10.3892/or.2015.3770

Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water 査読国際誌

Junichiro Futami, Haruna Fujiyama, Rie Kinoshita, Hidenori Nonomura, Tomoko Honjo, Hiroko Tada, Hirokazu Matsushita, Yoshito Abe, Kazuhiro Kakimi

PLOS ONE 9 ( 11 ) e113295-e113295 2014年11月

記述言語：英語出版者・発行元：PUBLIC LIBRARY SCIENCE

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.

DOI： 10.1371/journal.pone.0113295

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes 査読国際誌

Masakiyo Sakaguchi, Hitoshi Murata, Yumi Aoyama, Toshihiko Hibino, Endy Widya Putranto, I. Made Winarsa Ruma, Yusuke Inoue, Yoshihiko Sakaguchi, Ken-ichi Yamamoto, Rie Kinoshita, Junichiro Futami, Ken Kataoka, Keiji Iwatsuki, Nam-ho Huh

JOURNAL OF BIOLOGICAL CHEMISTRY 289 ( 34 ) 23389 - 23402 2014年8月

記述言語：英語出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Background: RAGE receptor plays a critical role in many inflammatory disorders. Results: Functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated cell survival. Conclusion: DAP10 membrane adaptor is critically involved in RAGE-mediated survival signaling upon S100A8/A9 binding. Significance: This is the first report demonstrating that RAGE-mediated survival signaling is critically regulated by DAP10 interaction.
The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

DOI： 10.1074/jbc.M114.573071

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene 査読国際誌

Masakiyo Sakaguchi, Masami Watanabe, Rie Kinoshita, Haruki Kaku, Hideo Ueki, Junichiro Futami, Hitoshi Murata, Yusuke Inoue, Shun-Ai Li, Peng Huang, Endy Widya Putranto, I. Made Winarsa Ruma, Yasutomo Nasu, Hiromi Kumon, Nam-ho Huh

MOLECULAR BIOTECHNOLOGY 56 ( 7 ) 621 - 630 2014年7月

記述言語：英語出版者・発行元：HUMANA PRESS INC

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1 alpha, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

DOI： 10.1007/s12033-014-9738-0

A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector 査読国際誌

Masami Watanabe, Masakiyo Sakaguchi, Rie Kinoshita, Haruki Kaku, Yuichi Ariyoshi, Hideo Ueki, Ryuta Tanimoto, Shin Ebara, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Peng Huang, Yasutomo Nasu, Nam-Ho Huh, Hiromi Kumon

ONCOLOGY REPORTS 31 ( 3 ) 1089 - 1095 2014年3月

記述言語：英語出版者・発行元：SPANDIDOS PUBL LTD

Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

DOI： 10.3892/or.2013.2958

3P-014 タンパク質カチオン化技術を用いた高感度抗体検出技術の開発(タンパク質工学,一般講演)

木戸桃子, 藤田佳那, 勝瀬奈津美, 新土居奈緒美, 愛宕祐基, 木下理恵, 野々村英典, 本莊知子, 二見淳一郎

日本生物工学会大会講演要旨集 66 198 - 198 2014年

3P-020 高純度・水溶性ヒト全長Cancer/Testis抗原リソースの整備(タンパク質工学,一般講演)

藤田佳那, 木戸桃子, 新土居奈緒美, 勝瀬奈津美, 愛宕祐基, 木下理恵, 野々村英典, 本莊知子, 二見淳一郎

日本生物工学会大会講演要旨集 66 199 - 199 2014年

BIO-NANOCAPSULE-LIPOSOME CONJUGATES FOR IN VIVO PINPOINT DRUG AND GENE DELIVERY 査読国際誌

Takeshi Kasuya, Joohee Jung, Rie Kinoshita, Yasumasa Goh, Takashi Matsuzaki, Masumi Iijima, Nobuo Yoshimoto, Katsuyuki Tanizawa, Shun&apos, ichi Kuroda

METHODS IN ENZYMOLOGY; LIPOSOMES, PT F 464 147 - 166 2009年

記述言語：英語出版者・発行元：ELSEVIER ACADEMIC PRESS INC

A bio-nanocapsule (BNC) is an similar to 50-nm hepatitis B virus (HBV) subviral particle comprising HBV envelope L proteins and a lipid bilayer, and is synthesized in recombinant Saccharomyces cerevisiae. When BNCs are administered intravenously in a mouse xenograft model, they can accumulate specifically in human liver-derived tissues and enter cells efficiently by the HBV-derived human liver-specific infection machinery, localized at the outer-membrane pre-5 region of the L protein. BNC specificity for the human liver can be altered to other tissues by substituting the pre-S region using targeting molecules (e.g., antibodies, lectins, cytokines). BNCs can spontaneously form complexes with liposomes (LPs) by the membrane fusogenic activity of the pre-S region. LPs containing various therapeutic materials (e.g., chemicals, proteins, DNA, RNA) can therefore be covered with BNCs to form an similar to 150-nm BNC-LP conjugate. BNC-LP conjugates injected intravenously can deliver incorporated materials to target tissues specifically and efficiently by utilizing the HBV-derived infection machinery. The stability of BNC-LP conjugates in the blood circulation is similar to that of PEGylated LPs. In this chapter, we describe the preparation and in vivo application of BNC-LP conjugates, and the potential of BNC-LP conjugates as in vivo pinpoint drug delivery systems.

DOI： 10.1016/S0076-6879(09)64008-8

Detection of oligomerization and conformational changes in the Na(+)/H(+) antiporter from Helicobacter pylori by fluorescence resonance energy transfer 査読国際誌

A Karasawa, Y Tsuboi, H Inoue, R Kinoshita, N Nakamura, H Kanazawa

JOURNAL OF BIOLOGICAL CHEMISTRY 280 ( 51 ) 41900 - 41911 2005年12月

記述言語：英語出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Oligomerization and conformational changes in the Na(+)/H(+) antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na(+)/H(+) antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li(+) was found to cause a significant decrease in FRET regardless of the presence or absence of Delta pH across the membranes, whereas Na(+) caused a much weaker effect. This Li(+) effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp(141) to Asn mutation, which results in defective Li(+)/H(+) antiporter activity, possibly Li(+) binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li(+) binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.

DOI： 10.1074/jbc.M510795200