九州大学-研究者情報 [巣山慶太郎 (助教) 基幹教育院自然科学実験系部門]

巣山慶太郎（すやまけいたろう）

データ更新日：2023.09.27

助教　／　基幹教育院自然科学実験系部門生体分子化学研究室

原著論文

1.	Daiki Tatsubo, Keitaro Suyama, Naoki Sakamoto, Keisuke Tomohara, Suguru Taniguchi, Iori Maeda, Takeru Nose, Determining the sequence-dependency of self-assembly of elastin-like peptides using short peptide analogs with shuffled repetitive sequences , Biochemistry, https://doi.org/10.1021/acs.biochem.3c00146, 62, 17, 2559-2570, 2023.08.
2.	Kohei Yoshida, Keitaro Suyama, Shin Matsushita, Iori Maeda, Takeru Nose, Development of the efficient preparation method for thermoresponsive elastin‐like peptides using liquid‐phase synthesis combined with fragment condensation strategy, Journal of Peptide Science, 10.1002/psc.3528, e3528, 2023.06.
3.	Keitaro Suyama, Masayuki Murashima, Iori Maeda, Takeru Nose, Aromatic compounds scavenging ability of thermo-responsive short elastin-like peptide (FPGVG)n analogs., Peptide Science 2022, 25-26, 2023.03.
4.	Keitaro Suyama, Hitoshi Kesamaru, Takashi Okubo, Kazumi Kasatani, Keisuke Tomohara, Ayami Matsushima, Takeru Nose, High cytotoxicity of a degraded TBBPA, dibromobisphenol A, through apoptotic and necrosis pathways., Heliyon, 10.1016/j.heliyon.2023.e13003, 9, 1, e13003, 2023.01, Halogenated flame retardants comprising bisphenol A (BPA) derivatives, such as tetrabromobisphenol A (TBBPA), have been studied their adverse effects on human health. However, despite the fact that these halogenated BPAs are easily degraded in the environment, the risks to living organisms due to these degraded products have mostly been overlooked. To evaluate the potential toxicity of degraded TBBPAs and related compounds, we examined the cytotoxicity of halogenated bisphenol A derivatives possessing one to four halogen atoms in vitro. The results indicated that the degraded TBBPA derivatives exhibited strong cytotoxicity against HeLa cells than TBBPA. Interestingly, the di-halogenated BPA derivatives possessing two halogen atoms exhibited the strongest cytotoxicity among tested compounds. In addition, a lactate dehydrogenase release assay, fluorescence spectroscopy and flow cytometry results indicated that dibromo-BPA and diiodo-BPA induced both apoptotic and necrotic cell death by damaging the cell membranes of HeLa cells. Moreover, Escherichia coli growth was inhibited in the presence of dehalogenated TBBPA and related compounds. These findings suggest that halogenated BPA derivatives that leak from various flame-retardant-containing products require strict monitoring, as not only TBBPA but also its degraded products in environment can exert adverse effects to human health..
5.	Shogo Sumiyoshi, Keitaro Suyama, Naoki Tanaka, Takumi Andoh, Akihiko Nagata, Keisuke Tomohara, Suguru Taniguchi, Iori Maeda, Takeru Nose, Development of truncated elastin-like peptide analogues with improved temperature-response and self-assembling properties, Scientific Reports, 10.1038/s41598-022-23940-0, 12, 1, 2022.11, Abstract Functional peptides, which are composed of proteinogenic natural amino acids, are expected to be used as biomaterials with minimal environmental impact. Synthesizing a functional peptide with a shorter amino acid sequence while retaining its function is a easy and economical strategy. Furthermore, shortening functional peptides helps to elucidate the mechanism of their functional core region. Truncated elastin-like peptides (ELPs) are peptides consisting of repetitive sequences, derived from the elastic protein tropoelastin, that show the thermosensitive formation of coacervates. In this study, to obtain shortened ELP analogues, we synthesized several (Phe-Pro-Gly-Val-Gly)_n (FPGVG)_n analogues with one or two amino acid residues deleted from each repeat sequence, such as the peptide analogues consisting of FPGV and/or FPG sequences. Among the novel truncated ELP analogues, the 16-mer (FPGV)₄ exhibited a stronger coacervation ability than the 25-mer (FPGVG)₅. These results indicated that the coacervation ability of truncated ELPs was affected by the amino acid sequence and not by the peptide chain length. Based on this finding, we prepared Cd²⁺-binding sequence-conjugated ELP analogue, AADAAC-(FPGV)₄, and found that it could capture Cd²⁺. These results indicated that the 16-mer (FPGV)₄ only composed of proteinogenic amino acids could be a new biomaterial with low environmental impact..
6.	Naoki Tanaka, Keitaro Suyama, Keisuke Tomohara, Iori Maeda, Takeru Nose, Branched short elastin-like peptides with temperature responsiveness obtained by EDTA-mediated multimerization, JOURNAL OF PEPTIDE SCIENCE, 10.1002/psc.3449, 2022.09, Elastin-like peptides (ELPs) exhibit a reversible phase transition, known as coacervation, triggered by temperature changes. This property makes them useful as stimuli-responsive molecular materials for various applications. Among ELPs, short peptide chain lengths have some advantages over long peptide chain lengths because short ELPs can be easily obtained by chemical synthesis, allowing the use of various amino acids, including D-type and unnatural amino acids, at any position in the sequence. Moreover, the incorporated amino acids readily affect the temperature-responsive behavior of ELPs. However, to be utilized in various applications, it is necessary to develop short ELPs and to investigate their temperature-responsive properties. To obtain further insights into the temperature-responsive behavior of the short ELPs, we investigated branched short ELP analogs composed of (FPGVG)(n) chains (n = 1 or 2, abbreviated as F1 and F2, respectively). We synthesized multimers composed of four F1 chains or two to four F2 chains using ethylenediaminetetraacetic acid (EDTA) as a central component of multimerization. Our results show that the multimers obtained exhibited coacervation in aqueous solutions whereas linear F1 or F2 did not. Furthermore, the structural features of the obtained multimers were the same as those of linear (FPGVG)(4). In this study, we demonstrated that molecules capable of coacervation can be obtained by multimerization of F1 or F2. The temperature-responsive molecules obtained using short ELPs make it possible to use them as easy-to-synthesize peptide tags to confer temperature responsiveness to various molecules, which will aid the development of temperature-responsive biomaterials with a wide variety of functions..
7.	Keitaro Suyama, Marin Shimizu, Iori Maeda, Takeru Nose, Flexible customization of the self-assembling abilities of short elastin-like peptide Fn analogs by substituting N-terminal amino acids, BIOPOLYMERS, 10.1002/bip.23521, 2022.07, Elastin-like peptides (ELPs) are thermoresponsive biopolymers inspired by the characteristic repetitive sequences of natural elastin. As ELPs exhibit temperature-dependent reversible self-assembly, they are expected to be biocompatible thermoresponsive materials for drug delivery carriers. One of the most widely studied ELPs in this field is the repetitive pentapeptide, (VPGXG)(n). We previously reported that phenylalanine-containing ELP (Fn) analogs, in which the former Val residue of the repetitive sequence (VPGVG)(n) is replaced by Phe, show coacervation with a short chain length (n = 5). Owing to their short sequences, Fn analogs are easily modified in amino acid sequences via simple chemical synthesis, and are useful for investigating the relationship between peptide sequences and temperature responsiveness. In this study, we developed Fn analogs by replacing Phe residue(s) with other amino acids or introducing another amino acid at the N-terminus. The temperature responsiveness of the Fn analogs changed drastically with the substitution of a single Phe residue, suggesting that aromatic amino acids play an important role in their self-assembly. In addition, the self-assembling ability of Fn was enhanced by increasing the bulkiness of the N-terminal amino acids. Therefore, the N-terminal residue was considered to be important for hydrophobicity-induced intermolecular interactions between the peptides during coacervation..
8.	Suyama Keitaro、Sumiyoshi Shogo、Tanaka Naoki、Ando Takumi、Nagata Akihiko、Tomohara Keisuke、Taniguchi Suguru、Maeda Iori、Nose Takeru, Development of temperature-responsive short-chain peptide analogues based on elastin-like peptide FPGVG, Peptide Science 2021, 141-142, 2022.03.
9.	Tanaka Naoki、Suyama Keitaro、Tomohara Keisuke、Maeda Iori、Nose Takeru, Development of temperature-responsive peptides by EDTA-mediated multimerization of short (FPGVG)n chains, Peptide Science 2021, 83-84, 2022.03.
10.	Shogo Sumiyoshi, Keitaro Suyama, Daiki Tatsubo, Naoki Tanaka, Keisuke Tomohara, Suguru Taniguchi, Iori Maeda, Takeru Nose, Metal ion scavenging activity of elastin-like peptide analogues containing a cadmium ion binding sequence, SCIENTIFIC REPORTS, 10.1038/s41598-022-05695-w, 12, 1, 2022.02, The development of simple and safe methods for recovering environmental pollutants, such as heavy metals, is needed for sustainable environmental management. Short elastin-like peptide (ELP) analogues conjugated with metal chelating agents are considered to be useful as metal sequestering agents as they are readily produced, environment friendly, and the metal binding domain can be selected based on any target metal of interest. Due to the temperature dependent self-assembly of ELP, the peptide-based sequestering agents can be transformed from the solution state into the particles that chelate metal ions, which can then be collected as precipitates. In this study, we developed a peptide-based sequestering agent, AADAAC-(FPGVG)(4), by introducing the metal-binding sequence AADAAC on the N-terminus of a short ELP, (FPGVG)(4). In turbidity measurements, AADAAC-(FPGVG)(4) revealed strong self-assembling ability in the presence of metal ions such as Cd2+ and Zn2+. The results from colorimetric analysis indicated that AADAAC-(FPGVG)(4) could capture Cd2+ and Zn2+. Furthermore, AADAAC-(FPGVG)(4) that bound to metal ions could be readily recycled by treatment with acidic solution without compromising its metal binding affinity. The present study indicates that the fusion of the metal-binding sequence and ELP is a useful and powerful strategy to develop cost-effective heavy metal scavenging agents with low environmental impacts..
11.	Masaki Iwamoto, Takahiro Masuya, Mari Hosose, Koki Tagawa, Tomoka Ishibashi, Keitaro Suyama, Takeru Nose, Eiji Yoshihara, Michael Downes, Ronald M. Evans, Ayami Matsushima, Bisphenol A derivatives act as novel coactivator-binding inhibitors for estrogen receptor beta, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1016/j.jbc.2021.101173, 297, 5, 2021.11, Bisphenol A and its derivatives are recognized as endocrine disruptors based on their complex effects on estrogen receptor (ER) signaling. While the effects of bisphenol derivatives on ER alpha have been thoroughly evaluated, how these chemicals affect ER beta signaling is less well understood. Herein, we sought to identify novel ER beta ligands using a radioligand competitive binding assay to screen a chemical library of bisphenol derivatives. Many of the compounds identified showed intriguing dual activities as both ER alpha agonists and ER beta antagonists. Docking simulations of these compounds and ER beta suggested that they bound not only to the canonical binding site of ER beta but also to the coactivator binding site located on the surface of the receptor, suggesting that they act as coactivator-binding inhibitors (CBIs). Receptor-ligand binding experiments using WT and mutated ER beta support the presence of a second ligand-interaction position at the coactivator-binding site in ER beta, and direct binding experiments of ER beta and a coactivator peptide confirmed that these compounds act as CBIs. Our study is the first to propose that bisphenol derivatives act as CBIs, presenting critical insight for the future development of ER signaling-based drugs and their potential to function as endocrine disruptors..
12.	Keitaro Suyama, Marin Shimizu, Iori Maeda, Takeru Nose., Role of Phe residues in elastin-like peptide (FPGVG)₅ on self-assembly properties, Peptide Science 2020, 59-60, 2021.03.
13.	Shogo Sumiyoshi, Keitaro Suyama, Takeru Nose, Development of self-aggregating elastin-like peptide analogs with a metal-binding sequence, Peptide Science 2020, 67-68, 2021.03.
14.	Iori Maeda, Suguru Taniguchi, Yumi Moriuchi, Naruhiko Sawa, Asako Inoue, Tomoyuki Usa, Noriko Watanabe, Keitaro Suyama, Takeru Nose, Fractionation of water-soluble elastin from pig aorta by coacervation method, Peptide Science 2020, 65-66, 2021.03.
15.	Koki Tagawa, Keitaro Suyama, Hitoshi Kesamaru, Takahiro Masuya, Takeru Nose, Ayami Matsushima, Design and synthesis of a universal coactivator peptide binding to the estrogen receptor and NURR1, Peptide Science 2020, 123-124, 2021.03.
16.	Xiaohui Liu, Keitaro Suyama, Takeru Nose, Miki Shimohigashi, Yasuyuki Shimohigashi, Bisphenol-C is the strongest bifunctional ERα-agonist and ERβ-antagonist due to magnified halogen bonding, PLoS ONE, https://doi.org/ 10.1371/journal.pone.0246583, 16, 2, e0246583, 2021.02.
17.	Keitaro Suyama, Mika Mawatari, Daiki Tatsubo, Iori Maeda, Takeru Nose., Simple Regulation of the Self-Assembling Ability by Multimerization of Elastin-Derived Peptide (FPGVG)n Using Nitrilotriacetic Acid as a Building Block., ACS Omega, 10.1021/acsomega.0c06140. , 6, 8, 5705-5716, 2021.02.
18.	Wataru Iwasaki, Chiwa Kataoka, Kazuyuki Sawadaishi, Keitaro Suyama, Nobutomo Morita, Masaya Miyazaki , A Simple, Low Cost, Sensitive, and Portable Electrochemical Immunochromatography Sensing Device to Measure Estrone-3-Sulfate , Sensors , https://doi.org/10.3390/s20174781, 20, 17, 4781-4781, 2020.08.
19.	Keitaro Suyama, Shuhei Kaneko, Hitoshi Kesamaru, Xiaohui Liu, Ayami Matsushima, Yoshimitsu Kakuta, Takashi Okubo, Kazumi Kasatani, Takeru Nose, Evaluation of the Influence of Halogenation on the Binding of Bisphenol A to the Estrogen-Related Receptor γ, Chemical Research in Toxicology, 10.1021/acs.chemrestox.9b00379, 33, 4, 889-902, 2020.04, [URL], Halogenation of organic compounds is one the most important transformations in chemical synthesis and is used for the production of various industrial products. A variety of halogenated bisphenol analogs have recently been developed and are used as alternatives to bisphenol A (BPA), which is a raw material of polycarbonate that has adverse effects in animals. However, limited information is available on the potential toxicity of the halogenated BPA analogs. In the present study, to assess the latent toxicity of halogenated BPA analogs, we evaluated the binding and transcriptional activities of halogenated BPA analogs to the estrogen-related receptor γ(ERRÎ³), a nuclear receptor that contributes to the growth of nerves and sexual glands. Fluorinated BPA analogs demonstrated strong ERRγbinding potency, and inverse antagonistic activity, similar to BPA. X-ray crystallography and fragment molecular orbital (FMO) calculation revealed that a fluorine-substituted BPA analog could interact with several amino acid residues of ERRÎ³-LBD, strengthening the binding affinity of the analogs. The ERRγbinding affinity and transcriptional activity of the halogenated BPAs decreased with the increase in the size and number of halogen atom(s). The IC₅₀ values, determined by the competitive binding assay, correlated well with the binding energy obtained from the docking calculation, suggesting that the docking calculation could correctly estimate the ERRγbinding potency of the BPA analogs. These results confirmed that ERRγhas a ligand binding pocket that fits very well to BPA. Furthermore, this study showed that the binding affinity of the BPA analogs can be predicted by the docking calculation, indicating the importance of the calculation method in the risk assessment of halogenated compounds..
20.	Keitaro Suyama, Mika Mawatari, Daiki Tatsubo, Iori Maeda, Takeru Nose, Concentration Dependent Coacervation Property of Nonlinear Elastin-derived Peptide (FPGVG)n Analogs, Peptide Science 2019, 29-30, 2019, 29–30., 2020.03, Elastin-like peptide (ELP) analogs demonstrate reversible temperature-dependent association (coacervation) under physiological conditions. Previously, we revealed that simply dimerized and cyclized short ELP analogs show stronger coacervation activity than monomeric and linear counterpart of them, respectively. In this study, we investigated concentration dependency of the transition temperature of nonlinear short ELPs to estimate their coacervation activity. It was revealed that the transition temperature of nonlinear ELPs could be described as the logarithmic function of peptide concentration. In addition, by using this quantitative relationship, it was able to compare and estimate the coacervation activity of artificial ELPs with different water solubility..
21.	Naoki Sakamoto, Daiki Tatsubo, Keiji Sato, Keisuke Tomohara, Keitaro Suyama, Iori Maeda, Takeru Nose, Development of Liposome Like Nanostructures Composed of Short Amphiphilic Elastin-Like Peptides, Peptide Science 2019, 113-114, 2019, 113–114., 2020.03.
22.	Iori Maeda, Miki Kawakami, Suguru Taniguchi, Keitaro Suyama, Takeru Nose, Stability of Phe-containing Elastin-like Peptides against Proteases in Digestive Organs, Peptide Science 2019, 161-162, 2019, 161–162., 2020.03.
23.	Xiaohui Liu, Keitaro Suyama, Junichi Shiki, Kohei Torikai, Takeru Nose, Miki Shimohigashi, Yasuyuki Shimohigashi, Bisphenol AF Halogen bonding effect is a major driving force for the dual ERα-agonist and ERβ-antagonist activities, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2019.115274, 28, 3, 2020.02, [URL], 17β-Estradiol (E2) is a natural steroid ligand for the structurally and physiologically independent estrogen receptors (ERs) ERα and ERβ. We recently observed that CF₃-containing bisphenol AF (BPAF) works as an agonist for ERα but as an antagonist for ERβ. Similar results were also observed for the CCl₃-containing bisphenol designated as HPTE. Both BPAF and HPTE are comprised of a tri-halogenated methyl group in the central alkyl moiety of their bisphenol structures, which strongly suggests that halogens contribute directly to the agonist/antagonist dual biological functions. We conducted this study to investigate the structure-activity relationships by assessing together newly synthesized CF₃- and CBr₃-containing bisphenol E analogs (BPE-X). We first tested bisphenols for their receptor binding ability and then for their transcriptional activities. Halogen-containing bisphenols were found to be fully active for ERα, but almost completely inactive for ERβ. When we examined these bisphenols for their inhibitory activities for E2 in ERβ, we observed that they worked as distinct antagonists. The ascending order of agonist/antagonist dual biological functions was BPE-F
24.	Xiaohui Liu, Hiroki Sakai, Mitsuhiro Nishigori, Keitaro Suyama, Tasuku Nawaji, Shin Ikeda, Makoto Nishigouchi, Hiroyuki Okada, Ayami Matsushima, Takeru Nose, Miki Shimohigashi, Yasuyuki Shimohigashi, Receptor-binding affinities of bisphenol A and its next-generation analogs for human nuclear receptors, Toxicology and Applied Pharmacology, 10.1016/j.taap.2019.114610, 377, 2019.08, [URL], An endocrine-disrupting chemical Bisphenol A (BPA) binds specifically to a nuclear receptor (NR) named ERRγ. Although the importance of receptor-binding evaluation for human NRs is often stressed, the binding characteristics of so-called next-generation (NextGen) bisphenol compounds are still poorly understood. The ultimate objective of this investigation was to evaluate BPA and its NextGen analogs for their abilities to bind to 21 human NRs, the greatest members of NRs for which tritium-labeled specific ligands were available. After establishing the detailed assay conditions for each NR, the receptor binding affinities of total 11 bisphenols were evaluated in competitive binding assays. The results clearly revealed that BPA and the NextGen bisphenols of BPAF, BPAP, BPB, BPC, BPE, and BPZ were highly potent against one or more of NRs such as CAR, ERα, ERβ, ERRγ, and GR, with IC₅₀ values of 3.3–73 nM. These bisphenols were suggested strongly to be disruptive to these NRs. BPM and BPP also appeared to be disruptive, but less potently. BPF exhibited only weak effects and only against estrogen-related NRs. Surprisingly, most doubtful bisphenol BPS was supposed not to be disruptive. The NRs to which BPA and NextGen bisphenols did not bind were RARα, RARβ, RARγ, and VDR. PPARγ, RORα, RORβ, RORγ, RXRα, RXRβ, and RXRγ, exhibited very weak interaction with these bisphenols. The ten remaining NRs, namely, ERRγ, ERβ, ERα, CAR, GR, PXR, PR, AR, LXRβ, and LXRα, showed distinctly strong binding to some bisphenols in this order, being likely to have consequential endocrine-disruption effects..
25.	Xiaohui Liu, Hiroki Sakai, Mitsuhiro Nishigori, Keitaro Suyama, Tasuku Nawaji, Shin Ikeda, Makoto Nishigouchi, Hiroyuki Okada, Ayami Matsushima, Takeru Nose, Miki Shimohigashi, Yasuyuki Shimohigashi , Receptor-binding Affinities of Bisphenol A and Its Next-generation Analogs for Human Nuclear Receptors, Toxicology and Applied Pharmacology, https://doi.org/10.1016/j.taap.2019.114610, in press, 2019.06, An endocrine-disrupting chemical Bisphenol A (BPA) binds specifically to a nuclear receptor (NR) named ERRγ. Although the importance of receptor-binding evaluation for human NRs is often stressed, the binding characteristics of so-called next-generation (NextGen) bisphenol compounds are still poorly understood. The ultimate objective of this investigation was to evaluate BPA and its NextGen analogs for their abilities to bind to 21 human NRs, the greatest members of NRs for which tritium-labeled specific ligands were available. After establishing the detailed assay conditions for each NR, the receptor binding affinities of total 11 bisphenols were evaluated in competitive binding assays. The results clearly revealed that BPA and the NextGen bisphenols of BPAF, BPAP, BPB, BPC, BPE, and BPZ were highly potent against one or more of NRs such as CAR, ERα, ERβ, ERRγ, and GR, with IC50 values of 3.3–73 nM. These bisphenols were suggested strongly to be disruptive to these NRs. BPM and BPP also appeared to be disruptive, but less potently. BPF exhibited only weak effects and only against estrogen-related NRs. Surprisingly, most doubtful bisphenol BPS was supposed not to be disruptive. The NRs to which BPA and NextGen bisphenols did not bind were RARα, RARβ, RARγ, and VDR. PPARγ, RORα, RORβ, RORγ, RXRα, RXRβ, and RXRγ, exhibited very weak interaction with these bisphenols. The ten remaining NRs, namely, ERRγ, ERβ, ERα, CAR, GR, PXR, PR, AR, LXRβ, and LXRα, showed distinctly strong binding to some bisphenols in this order, being likely to have consequential endocrine-disruption effects..
26.	Hitoshi Kesamaru, Keitaro Suyama, Takeru Nose, Importance of Receptor Conformations in Docking Calculation-Based Risk Assessment for Endocrine Disruptors against Estrogen Receptor α, ACS Omega, 10.1021/acsomega.9b00050, 4, 4, 6620-6629, 2019.04, [URL], Employment of appropriate receptor conformations as templates is essential for appropriate identification of the latent receptor binding ability of chemicals using in silico docking calculations. In this study, we performed docking calculations using a number of agonist- and antagonist-bound conformations of 83 estrogen receptor (ER) α-ligand-binding domains as templates to clarify the type of receptor conformations required for reasonable identification of endocrine disruptors. Our results showed that 17β-estradiol and diethylstilbestrol (ERα agonists) bound preferentially to the agonist conformations, whereas 4-hydroxytamoxifen and raloxifene (ERα antagonists) bound selectively to the antagonist conformations. We also observed that bisphenol A analogues, which are partial agonists, bound more moderately and preferentially to the agonist conformations as compared with the antagonist conformations. Additionally, the docking calculations were able to estimate biological agonist or antagonist activity of chemicals based on the receptor conformation selectivity. Furthermore, structural analyses of the ligand-binding domains and docking calculation utilizing C-terminal-truncated receptors indicated that the C-terminal regions of these domains were capable of discriminating agonists from nonagonists. These results suggest that both agonist- and antagonist-binding conformations of receptors are necessary to predict the binding affinity and biological activity of chemicals for docking calculation-based risk assessment. Furthermore, this in silico method can be beneficial for drug discovery because it is useful for rapid searching of ligands for receptors and preventing the side effects caused by unfavorable receptor binding..
27.	Keitaro Suyama, Daiki Tatsubo, Suguru Taniguchi, Iori Maeda, Takeru Nose, Development of Self-assembling Short Elastin-derived Peptide Analogs: Linear and Nonlinear (FPGVG)n Analogs, Peptide Science 2018, 117-117, 2018, 117., 2019.03, The elastin-derived peptide analog, (FPGVG)n, demonstrates temperature-dependent self-association (coacervation) property. In this study, we revealed that simple dimerization and cyclization of (FPGVG)n analogs induced strong coacervation ability compared to the ability of monomeric linear (FPGVG)5..
28.	Daiki Tatsubo, Keitaro Suyama, Iori Maeda, Takeru Nose, Structure and Function of the Elastin-like Short Peptide Analogs with Shuffled Sequences Based on (FPGVG)5., Peptide Science 2018: Proceedings of the 10th International Peptide Symposium, 65-65, 2019.03.
29.	Keisuke Tomohara, Isao Adachi, Yoshikazu Horino, Hitoshi Kesamaru, Hitoshi Abe, Keitaro Suyama, Takeru Nose, DMSO-Perturbing Assay for Identifying Promiscuous Enzyme Inhibitors, ACS Medicinal Chemistry Letters, 10.1021/acsmedchemlett.9b00093, 10, 6, 923-928, 2019.01, [URL], In search for enzyme inhibitors, we often encounter "promiscuous" enzyme inhibitors exhibiting nonspecific binding property toward enzyme active site. Therefore, inhibitory candidates should be mechanistically characterized as early as possible in discovery processes. However, there remains a lack of highly reliable and readily available methodology to evaluate specificity of initial hits inhibitors. The present study developed and established a novel DMSO-perturbing assay to identify promiscuous enzyme inhibitors. The assay successfully identified nonspecific binding inhibitors with a broad scope, typically by the attenuation of inhibitory activity by the influence of DMSO-addition. This attenuation would be attributed to the nonspecific binding property of inhibitors toward both productive and nonproductive (nondenatured) states of enzymes in perturbation solution. This working hypothesis was supported by spectroscopic analyses of enzyme conformations and analyses of solvent effects on perturbation. Overall, these results provided a novel concept of the DMSO-perturbing assay..
30.	Daiki Tatsubo, Keitaro Suyama, Masaya Miyazaki, Iori Maeda, and Takeru Nose, Stepwise Mechanism of Temperature-Dependent Coacervation of the Elastin-like Peptide Analogue Dimer, (C(WPGVG)3)2, Biochemistry, 10.1021/acs.biochem.7b01144, 57, 10, 1582-1590, 2018.06, Elastin-like peptides (ELPs) are distinct, repetitive, hydrophobic sequences, such as (VPGVG)n, that exhibit coacervation, the property of reversible, temperature-dependent self-association and dissociation. ELPs can be found in elastin and have been developed as new scaffold biomaterials. However, the detailed relationship between their amino acid sequences and coacervation properties remains obscure because of the structural flexibility of ELPs. In this study, we synthesized a novel, dimeric ELP analogue (H-C(WPGVG)3-NH2)2, henceforth abbreviated (CW3)2, and analyzed its self-assembly properties and structural factors as indicators of coacervation. Turbidity measurements showed that (CW3)2 demonstrated coacervation at a concentration much lower than that of its monomeric form and another ELP. In addition, the coacervate held water-soluble dye molecules. Thus, potent and distinct coacervation was obtained with a remarkably short sequence of (CW3)2. Furthermore, fluorescence microscopy, dynamic light scattering, and optical microscopy revealed that the coacervation of (CW3)2 was a stepwise process. The structural factors of (CW3)2 were analyzed by molecular dynamics simulations and circular dichroism spectroscopy. These measurements indicated that helical structures primarily consisting of proline and glycine became more disordered at high temperatures with concurrent, significant exposure of their hydrophobic surfaces. This extreme change in the hydrophobic surface contributes to the potent coacervation observed for (CW3)2. These results provide important insights into more efficient applications of ELPs and their analogues, as well as the coacervation mechanisms of ELP and elastin..
31.	Keitaro Suyama, Daiki Tatsubo, Wataru Iwasaki, Masaya Miyazaki, Yuhei Kiyota, Ichiro Takahashi, Iori Maeda, Takeru Nose, Enhancement of self-aggregation properties of linear elastin-derived short peptides by simple cyclization strong self-aggregation properties of cyclo[FPGVG]_n, consisting only of natural amino acids, Biomacromolecules, 10.1021/acs.biomac.8b00353, 19, 8, 3201-3211, 2018.06, [URL], Elastin-like peptides (ELP) consist of distinctive repetitive sequences, such as (VPGVG)_n, exhibit temperature-dependent reversible self-assembly (coacervation), and have been considered to be useful for the development of thermo-responsive materials. Further fundamental studies evaluating coacervative properties of novel nonlinear ELPs could present design concepts for new thermo-responsive materials. In this study, we prepared novel ELPs, cyclic (FPGVG)_n (cyclo[FPGVG]_n, n = 1-5), and analyzed its self-assembly properties and structural characteristics. Cyclo[FPGVG]_n (n = 3-5) demonstrated stronger coacervation capacity than the corresponding linear peptides. The coacervate of cyclo[FPGVG]₅ was able to retain water-soluble dye molecules at 40°C, which implied that cyclo[FPGVG]₅ could be employed as a base material of DDS (Drug Delivery System) matrices and other biomaterials. The results of molecular dynamics simulations and circular dichroism measurements suggested that a certain chain length was required for cyclo[FPGVG]_n to demonstrate alterations in molecular structure that were critical to the exhibition of coacervation..
32.	Hujun Li, Asako Inoue, Suguru Taniguchi, Tomohiko Yukutake, Keitaro Suyama, Takeru Nose, Iori Maeda, Multifunctional biological activities of water extract of housefly larvae (Musca domestica), PharmaNutrition, 10.1016/j.phanu.2017.09.001, 5, 4, 119-126, 2017.12, [URL], Many types of insects have been used as foods and protein sources. In this study, we investigated the usefulness of housefly larvae (Musca domestica) based on their amino acid composition and multifunctional biological activities. First, the utility of the amino acid composition of housefly larvae was evaluated by amino acid analysis. Notably, the housefly larvae contained sufficient amounts of all essential amino acids, and the amino acid composition was similar to that of hen eggs. Second, we prepared housefly larvae water extract (HLWE) using the decoction method and explored the biological activities of the extract for potential application of the extract as a functional food. HLWE showed significant antioxidant activity (75.4% at 5.00 mg/mL), angiotensin-I-converting enzyme (ACE) inhibitory activity (half-maximal inhibitory concentration [IC ₅₀ ] = 0.430 mg/mL), and dipeptidyl peptidase-IV (DPP-IV) inhibitory activity ([IC ₅₀ ] = 3.52 mg/mL). We found that the low-molecular-weight constituents (6 kDa) contributed to DPP-IV inhibition. Our results suggested that housefly larvae may provide a useful source of multifunctional protein..
33.	Keitaro Suyama, Daiki Tatsubo, Hitoshi Kesamaru, Iori Maeda, Takeru Nose, Coacervation Property and Structural Analysis of Cyclic Analogs of Elastin-derived Peptide (FPGVG)n, Peptide Science 2016, 101-102, 2017.03, The elastin-derived peptide analog, (FPGVG)5, demonstrates a temperature- dependent self-association (coacervation) property. In this study, we prepared cyclic (FPGVG)n (n=1-5) analogs and investigated their coacervation and structural properties to assess their potential for use as a biomaterial. We found that the cyclic (FPGVG)n (n=3-5) analogs exhibited high coacervation ability. The results from the molecular dynamics simulation suggest that turn structures were important for coacervation of elastin-derived cyclic peptide analogs..
34.	Keitaro Suyama, Hitoshi Kesamaru, Daiki Tatsubo, Takeru Nose, Coacervation Properties and Structural Analysis of Aminobenzoyl-labeled Fluorescent Elastin-derived Peptides, Peptide Science 2015, 293-294, 2016.03, The elastin-derived peptide analog, (FPGVG)5, demonstrates a temperature- dependent self-association (coacervation) property. In this study, we prepared fluorescent-labeled (FPGVG)5 analogs, and evaluated their coacervation and fluorescent properties in order to assess their potential for use as a base material for fluorescent thermometers. We found that the fluorescent-labeled (FPGVG)5 analogs exhibited high coacervation ability. Results from the molecular dynamics calculation suggest that the fluorescent group is exposed on the surface of the molecule..
35.	Daiki Tatsubo, Keitaro Suyama, Takeru Nose, Fluorescence Analysis Using a Molecular Probe 1,8-ANS for Elucidation of the Molecular Mechanisms Underlying Coacervation of a Tryptophan-containing Elastin derived Dimeric Peptide, Peptide Science 2015, 95-96, 2016.03, A tryptophan-containing elastin-derived dimeric peptide (C(WPGVG)3)2 shows potent self-assembly activity. To elucidate the underlying coacervation mechanisms, we measured the fluorescence of (C(WPGVG)3)2 by using 1,8-ANS as a fluorescent probe. The results revealed that the peptide forms microaggregates at temperature and concentration lower than those at which visible coacervation occurs. These findings suggest that microaggregate formation is unrelated to the maturation of the coacervate drop, which is temperature-dependent..
36.	Keitaro Suyama, Suguru Taniguchi, Daiki Tatsubo, Iori Maeda, Takeru Nose, Dimerization effects on coacervation property of an elastin-derived synthetic peptide (FPGVG)5, Journal of Peptide Science, 10.1002/psc.2876, in press, 2016.03, A series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were synthesized. The new dimeric peptides showed significantly high coacervation ability compared to known elastin-derived peptide analogs. The molecular dynamics calculation results reveal that the dimeric peptides contain characteristic sheet-turnsheet motif involving a type II β-turn-like structure and form globular conformation..
37.	巣山慶太郎, 田坪大来, 谷口　卓, 袈裟丸仁志, 前田衣織, 野瀬健, Coacervation property and structural analysis of synthetic dimer peptides of aromatic amino acid containing elastin-derived peptides., Peptide Science 2014, 323-324, 2015.03, It has been shown that elastin-derived peptides containing aromatic amino acids, (FPGVG)n and (WPGVG)n, demonstrate coacervation at small repetition numbers (n = 3–5). In this study, we prepared dimer peptides of (FPGVG)5 and (WPGVG)3 and evaluated their coacervation properties. The coacervation ability of the peptides was significantly enhanced by the dimerization. The results of molecular dynamics calculation suggest that the sheet-turn-sheet motif is important for the self-assembly of FPGVG-related peptides..
38.	巣山慶太郎, 谷口卓, 前田衣織, 田坪大来, 野瀬健, Coacervation Property and Secondary Structure of Synthetic Dimer Peptides of Elastin-derived Pentapeptide Repeats , Peptide Science 2013, 277-278, 2014.03, Elastin is the core protein of elastic fibers in the elastic tissues, such as arterial walls, lungs, and skin, and plays an essential role in tissue biomechanics, providing the extensibility and condensability. Elastin shows self-association, which is referred as coacervation, under physiological conditions. Coacervation property of elastin characterized by reversible association/dissociation phenomena is considered to be important for its elasticity. Tropoelastin, the precursor protein of elastin, contains characteristic repetitive sequences in its hydrophobic regions. The pentapeptide sequence, Val-Pro-Gly-Val-Gly (VPGVG), is one of the repeating sequences, and presents in almost all mammal species analyzed so far. To clarify the mechanism of elastin’s elasticity, many studies about coacervation properties of elastin-derived peptide-analogs have been conducted. Many previous studies reported that large molecular size was needed for coacervation of elastin-derived peptides. For instance, (VPGVG)n demonstrated the coacervation property only when they had large numbers of repetition (n>40). Recently, we developed the synthetic hydrophobic oligomers (IPGVG)n and (FPGVG)n, and reported that they demonstrate coacervation at significantly smaller repetition numbers (n=5~7). Because of their low molecular size, these analogs are expected to be useful as model peptides for structure analysis and base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. In this study, to obtain more short coacervatable peptides, we synthesized dimers of pentapeptide (FPGVG)5 and investigated their coacervation property and secondary structures. To produce dimer peptides, cysteine-conjugated pentapeptide Cys-(FPGVG)5 and (FPGVG)5-Cys were synthesized by the solid-phase method using Fmoc strategy. By aerobic oxidation reaction of each Cys-peptide, homodimers, linked by disulfide bond, were obtained. Synthesized peptides were purified by HPLC and analyzed by MALDI-TOF- MS. The dimer peptides clearly showed coacervation around 15˚C in the condition at 10 mg/ml, whereas (FPGVG)5 required rather higher temperature and at least 20 mg/ml of peptide to demonstrate coacervation. These results indicated that the dimerization of elastin-derived peptide-analogs significantly enhanced the coacervation ability of (FPGVG)5. Furthermore, these dimer peptides showed reversible temperature-dependent conformation change in CD measurement..
39.	Kanako Nishio, Hirokazu Nishimura, Keitaro Suyama, Takeru Nose, Yasuyuki Shimohigashi, Halogenated Phe-containing endomorphin-2 analogs with mixed agonist and antagonist activities, Peptide Science 2012, in press, 2013.03.
40.	Yumi Kramitsu, Hirokazu Nishimura, Ryo Nakamura, Keitaro Suyama, Kazuhiro Matsumoto, Takeru Nose, Yasuyuki Shimohigashi, High-precision binding assay procedure of tachykinin receptor NK-1 for highly potent substance P analogs, Peptide Science 2012, in press, 2013.03.
41.	Ryo Nakamura, Hirokazu Nishimura, Keitaro Suyama, Takeru Nose, Yasuyuki Shimohigashi, The Effect of Halogenation of Phe-Phenyl Group of Two Consecutive Phe Residues Present in Neuropeptide Substance P on Its Specific Receptor Interaction, Peptide Science 2011, 157-158, 2012.03.
42.	Kanako Nishio, Hirokazu Nishimura, Keitaro Suyama, Yoshinori Abe, Takeru Nose, Ayami Matsushima, Yasuyuki Shimohigashi, Effects of the Halogenation of Phe-Phenyl Group of Two Consecutive Residues in Endomorphin-2 on the Interaction with the μ-Opioid Receptors, Peptide Science 2011, 171-172, 2012.03.
43.	Keitaro Suyama, Yoshifumi Sakai, Kazuhiro Matsumoto, Tsutomu Katsuki, Highly Enantioselective Hydrophosphonylation of Aldehydes: Base-Enhanced Aluminum-salalen Catalysis, Angewante Chemie, International Edition, 10.1002/anie.200905158, 49, 797-799, 2010.01.
44.	Keitaro Suyama, Kazuhiro Matsumoto, Tsutomu Katsuki, Asymmetric Lewis Acid Catalysis of Aluminum(salalen) Complexes: Friedel-Crafts Reaction of Indole, Heterocycles, 10.3987/COM-08-S(F)93, 77, 817-824, 2009.02.

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