Papers - USUI Kenji
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Nanomolar Aggregation and Neurotoxicity of Amyloid beta Peptides with Site-Specific Modification of Oxidized Cholesterol Reviewed International coauthorship
K. Usui, J. D. Hulleman, J. F. Paulsson, S. J. Siegel, E. T. Powers, J. W. Kelly
Peptide Science 2010 2010 169 - 169 2011
Joint Work
Authorship：Lead author
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A Novel Nitric Oxide Sensor Using Fluorescent Peptides Attached to Iron Complexes Reviewed
Hiroshi Miyazaki, Kenji Usui, Satoshi Fujii
Peptides 2011 2011 224 - 255 2011
Joint Work
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A PNA Peptide Inducing DNAs to Form G-Quadruplex Structures Depending on a Protease Activity Reviewed
Kenji Usui, Keita Kobayashi, Naoki Sugimoto
Peptides 2011 2011 152 - 153 2011
Joint Work
Authorship：Lead author
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Construction of Detection System for Amyloid beta Peptide Localization and Aggregation Using Fluorescent and Luminescent Fusion Proteins Reviewed
Kenji Usui, Masayasu Mie, Takashi Andou, Naoki Sugimoto, Hisakazu Mihara, Eiry Kobatake
Peptide Science 2009 2009 111 - 112 2010
Joint Work
Authorship：Lead author
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Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity Reviewed International coauthorship
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 2009.11
Publisher：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 Reviewed
Kenji Usui, Kin-Ya Tomizaki, Hisakazu Mihara
Methods in Molecular Biology 570 273 - 284 2009
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 Reviewed
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 17 ( 1 ) 167 - 171 2007.1
Publisher：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 Reviewed
Sinya Watanabe, Kin-ya Tomizaki, Tsuyoshi Takahashi, Kenji Usui, Kotaro Kajikawa, Hisakazu Mihara
BIOPOLYMERS 88 ( 2 ) 131 - 140 2007
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Protein-fingerprint data mining of a designed alpha-helical peptide array Reviewed
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
MOLECULAR BIOSYSTEMS 2 ( 9 ) 417 - 420 2006.9
Publisher：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 Reviewed
K Usui, K Tomizaki, T Ohyama, K Nokihara, H Mihara
MOLECULAR BIOSYSTEMS 2 ( 2 ) 113 - 121 2006.2
Publisher：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 Reviewed
S Sano, KY Tomizaki, K Usui, H Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 16 ( 3 ) 503 - 506 2006.2
Publisher：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 Reviewed
Kenji Usui, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 731 - + 2006
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High throughput preparation of peptide arrays containing two fluorescent byes focusing on practical protein detection systems Reviewed
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Yasuo Oka, Kenji Usui, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 738 - + 2006
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Anomalous reflection of gold applicable for a practical protein-detecting chip platform Reviewed
S Watanabe, K Usui, K Tomizaki, K Kajikawa, H Mihara
MOLECULAR BIOSYSTEMS 1 ( 5-6 ) 363 - 365 2005.12
Publisher：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 2005.11
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 Reviewed
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 2005.11
Publisher：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 Reviewed
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 2005.10
Publisher：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 Reviewed
Kin-Ya Tomizaki, Kenji Usui, Hisakazu Mihara
ChemBioChem 6 ( 6 ) 782 - 799 2005.5
Joint Work
The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry wilt provide simple production protocols and specific orientations of capture agents on the -microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery. © 2005 Wiley-VCH Verlag GmbH &
Co. KGaA. -
Studies on Fluorescent Dyes and Surface Chemistry focusing on Practical Peptide Array Preparation Reviewed
NOKIHARA Kiyoshi, USUI Kenji, YONEMURA Koichi, OHYAMA Takafumi, OKA Yasuo, TOMIZAKI Kin‐ya, MIHARA Hisakazu
Pept Sci 2004 145 - 148 2005.3
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Optimization of peptide arrays on chip as novel practical protein characterization system Reviewed
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
Peptides 2004, Proceedings 176 - 177 2005