論文 - 臼井 健二
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Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity 査読あり 国際共著
Kenji Usui, John D. Hulleman, Johan F. Paulsson, Sarah J. Siegel, Evan T. Powers, Jeffery W. Kelly
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 ( 44 ) 18563 - 18568 2009年11月
出版者・発行元:NATL ACAD SCIENCES
Accumulation of amyloid beta-peptide (A beta) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between A beta and the aldehyde-bearing cholesterol oxidation product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase A beta amyloidogenicity. Here, we synthesized A beta variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified A beta formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from A beta Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified A beta under identical conditions and at the same concentration. Our results show that A beta modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of A beta.
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A designed peptide chip: Protein fingerprinting technology with a dry peptide array and statistical data mining 査読あり
Kenji Usui, Kin-Ya Tomizaki, Hisakazu Mihara
Methods in Molecular Biology 570 273 - 284 2009年
There has recently been increased interest in the potential for microarray technologies to study protein networks in a whole cell system within a single experiment. Protein-detecting microarrays are composed of numerous agents immobilized within a tiny area on solid surfaces to capture targeted proteins and to detect interactions in a high-throughput fashion. In this chapter, in order to extend the usability of peptide microarrays, we describe a novel dry peptide microarray format to obtain protein fingerprint (PFP) data sets and a statistical PFP data manipulation technique to quantitatively analyze targeted proteins. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.
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Screening of alpha-helical peptide ligands controlling a calcineurin-phosphatase activity 査読あり
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 17 ( 1 ) 167 - 171 2007年1月
出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD
In this paper, we describe an application of 202-membered fluorescently labeled peptide library designed to take an alpha-helix secondary structure. As a proof-of-concept experiment, a calmodulin (CaM)/calcineurin (Cn) pair was chosen to screen alpha-helical peptide ligands that tightly bind to CaM and also control enzymatic functions of Cn. Three peptides were successfully selected from the library by assaying Cn-phosphatase activities and peptide-CaM interactions (dual check process). The strategy using a designed peptide. library shows real promise as a peptide-based high-throughput screening system. (c) 2006 Elsevier Ltd. All rights reserved.
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Interactions between Peptides Containing Nucleobase Amino Acids and T7 Phages Displaying S. Cerevisiae Proteins 査読あり
S. Watanabe, K.-Y. Tomizaki, T. Takahashi, K. Usui, K. Kajikawa and H. Mihara
Biopolymers 88 ( 2 ) 131 - 140 2007年
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Protein-fingerprint data mining of a designed alpha-helical peptide array 査読あり
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
MOLECULAR BIOSYSTEMS 2 ( 9 ) 417 - 420 2006年9月
出版者・発行元:ROYAL SOC CHEMISTRY
The data generated from protein fingerprints with an a-helical peptide array were analyzed using several statistical methods such as hierarchical clustering analysis and principal component analysis to discriminate target proteins. The data generated from protein fingerprints with an a-helical peptide array were analyzed using several statistical methods such as hierarchical clustering analysis and principal component analysis to discriminate target proteins.
DOI: 10.1039/b608875a
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A novel peptide microarray for protein detection and analysis utilizing a dry peptide array system 査読あり
K Usui, K Tomizaki, T Ohyama, K Nokihara, H Mihara
MOLECULAR BIOSYSTEMS 2 ( 2 ) 113 - 121 2006年2月
出版者・発行元:ROYAL SOC CHEMISTRY
A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure tinder dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-Of-Use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an a-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These Studies imply that the dry peptide array system is a promising too] for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies.
DOI: 10.1039/b514263r
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A PNA-DNA hybridization chip approach for the detection of beta-secretase activity 査読あり
S Sano, KY Tomizaki, K Usui, H Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 16 ( 3 ) 503 - 506 2006年2月
出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD
Developed was the addressable chip technology based on the PNA-DNA complementary hybridization equipped with short seven-mer PNA-encoded peptides that can be a versatile scaffold to monitor on-chip immunoassays. We also developed and validated a methodology to perform beta-secretase enzyme assay with a highly sensitive fashion, resulting that a peptide substrate tethering dual fluorescent probes allowed us to detect beta-secretase activity 10 times more sensitively than assays in solution. (c) 2005 Elsevier Ltd. All rights reserved.
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Designed peptide microarrays for protein detection and characterization 査読あり
Kenji Usui, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 731 - + 2006年
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High throughput preparation of peptide arrays containing two fluorescent byes focusing on practical protein detection systems 査読あり
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Yasuo Oka, Kenji Usui, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 738 - + 2006年
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Anomalous reflection of gold applicable for a practical protein-detecting chip platform 査読あり
S Watanabe, K Usui, K Tomizaki, K Kajikawa, H Mihara
MOLECULAR BIOSYSTEMS 1 ( 5-6 ) 363 - 365 2005年12月
出版者・発行元:ROYAL SOC CHEMISTRY
A simple, convenient and label-free fiber optic detection system based on the characteristic property, 'anomalous reflection (AR)' of gold was developed and preliminary experiments showed that the AR signals were sensitive enough to monitor protein-peptide interactions on solid surfaces.
DOI: 10.1039/b513075c
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Kenji Usui, Tetsunori Ojima, Kin-Ya Tomizaki, Hisakazu Mihara
Nanobiotechnology 1 ( 2 ) 191 - 199 2005年11月
For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as "protein fingerprints" (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies. Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved.
DOI: 10.1385/NBT:1:2:191
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Difference in self-assembling morphology of peptide nanorings 査読あり
H Okamoto, T Yamada, H Miyazaki, T Nakanishi, K Takeda, K Usui, Obataya, I, H Mihara, H Azehara, W Mizutani, K Hashimoto, H Yamaguchi, Y Hirayama
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 ( 11 ) 8240 - 8248 2005年11月
出版者・発行元:JAPAN SOC APPLIED PHYSICS
We synthesized the peptide nanorings of cyclo[-(D-Ala-L-Gln)(3)], cyclo[-(D-CyS-L-Gln)(3)], CyC10[-D-Cys-L-HiS-D-Ala-L-Asn-Gly-L-Gln-1 and Cyc1o[-(L-Gln)(5)], and studied the way in which the difference in the type and/or number of component amino acid residues changes the self-assembling morphology of the nanorings on gold substrates by atomic force microscopy. The study revealed that CyClo[-(D-Ala-L-Gln)(3)] formed nanotube bundles through inter-ring hydrogen bonds, while the nanorings of CyC10[-(D-CyS-L-Gln)3] adhered to the gold surface directly due to the high affinity of thiol to gold. In contrast, a random amino acid sequence of cyclo[-D-CyS-L-HiS-D-Ala-L-Asn-GlY-L-Gln-] resulted in many isolated nanotubes, which were first observed in the present study. While the D,L-peptide nanotubes have very straight forms, the homo-L-peptide of cyclo[-(L-Gln)(5)] formed interesting randomly branching nanotubes that were entwined and grew on the substrate. Scanning tunneling microscopy was also performed and high-resolution images of both the peptide nanotubes and the nanotube bundles were obtained.
DOI: 10.1143/JJAP.44.8240
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IR study on stacking manner of peptide nanorings in peptide nanotubes 査読あり
Y Nagai, T Nakanishi, H Okamoto, K Takeda, Y Furukawa, K Usui, H Mihara
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 ( 10 ) 7654 - 7661 2005年10月
出版者・発行元:JAPAN SOC APPLIED PHYSICS
We here report our theoretical as well as experimental studies on the stacking manner of peptide nanorings (PNRs) in peptide nanotubes (PNTs). We focus on the molecular vibrations of N-H and C=O stretching modes and discuss this subject via their infrared (IR) spectroscopy, because PNTs are formed by the inter-ring H bonds between the adjacent PNRs via -N-H(...)O=C-. Symmetry analysis based on group theory reveals that parallel stacking causes two IR active modes in these molecular vibrations while three modes are active in the antiparallel stacking. This difference in the number of IR active modes is determined only by the stacking manner and not by the number of amino acid residues composing the PNRs. By using two typical PNRs Of cyclo[-((L)-Gln-D-Ala)(3)] and cyclo[-((L)-Gln-(D)-Ala)(4)], we further studied the favorable stacking manners of PNRs via IR observation. Our IR experiments as well as the ab initio energetics show that the former PNRs create a PNT by stacking themselves in parallel while the latter PNRs do so by stacking themselves in an antiparallel manner.
DOI: 10.1143/JJAP.44.7654
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Protein-Detecting Microarrays: Current Accomplishments and Requirements 査読あり
Kin-ya Tomizaki, Kenji Usui, Hisakazu Mihara
ChemBioChem 6 ( 6 ) 782 - 799 2005年5月
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Studies on Fluorescent Dyes and Surface Chemistry focusing on Practical Peptide Array Preparation 査読あり
NOKIHARA Kiyoshi, USUI Kenji, YONEMURA Koichi, OHYAMA Takafumi, OKA Yasuo, TOMIZAKI Kin‐ya, MIHARA Hisakazu
Pept Sci 2004 145 - 148 2005年3月
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Optimization of peptide arrays on chip as novel practical protein characterization system 査読あり
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
Peptides 2004, Proceedings 176 - 177 2005年
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Protein-detection microarrays using structure-based designed peptide libraries 査読あり
Kenji Usui, Tetsunori Ojima, Masato Suzuki, Sin-ya Watanabe, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
Peptides 2004, Proceedings 401 - 402 2005年
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Peptide Arrays with Designed Secondary Structures for Protein Characterization Using Fluorescent Fingerprint Patterns. 査読あり
臼井健二
Biopolymers ( 76 ) 129 - 139 2004年11月
単著
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Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns 査読あり
Kenji Usui, Mizuki Takahashi, Kiyoshi Nokihara, Hisakazu Mihara
MOLECULAR DIVERSITY 8 ( 3 ) 209 - 218 2004年9月
出版者・発行元:SPRINGER
A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.
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Development of a practical protein-chip using designed synthetic peptide-arrays 査読あり
K Nokihara, T Ohyama, K Usui, K Yonemura, K Tomizaki, H Mihara
KOBUNSHI RONBUNSHU 61 ( 10 ) 523 - 532 2004年
出版者・発行元:SOC POLYMER SCIENCE JAPAN
Novel high-throughput technologies, which can replace conventional electrophoresis and mass spectroscopic analyses, are in great demand for understanding the structure-function relationships of proteins. For a practical protein-detection system, arrays with immobilized designed synthetic peptides have been constructed. Peptides were labeled with fluorescent dyes in order to achieve high sensitivity. Two different types of prototype-arrayer have been constructed: one has a micro-dispensing system and the other a spot-printing system. Combinatorial peptide libraries, which consisted of beta-loop, beta-strand and alpha-helical peptides, were constructed by improved highly efficient solid-phase synthesis. Labeled peptides were covalently immobilized on solid supports such as precision glass plates. Various proteins were characterized with these peptide-arrays using a fluorescent scanner to give the "protein fingerprint" which is characteristic of the individual proteins. The results of the present prototype system demonstrate the practicality of protein-chips as a new generation of biochips.