Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy

Kitamura, Akira; Ishida, Yoshihito; Kubota, Hiroshi; Pack, Chan-Gi; Homma, Takayuki; Ito, Shinya; Araki, Kazutaka; Kinjo, Masataka; Nagata, Kazuhiro

Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy

Kitamura, Akira; Ishida, Yoshihito; Kubota, Hiroshi et al.

Biochemical and Biophysical Research Communications, 2018, 497(1), 279-284

Permalink : https://hdl.handle.net/2115/72752

このアイテムのアクセス数:843件（2026-10-13 23:03 集計）

閲覧可能ファイル

ファイル	フォーマット	サイズ	閲覧回数	説明
BBRC_20180210	pdf	539 KB	964

論文情報

ファイル出力 EndNote Basic出力 Mendeley出力

アクセス権	open access
URI	https://hdl.handle.net/2115/72752
タイトル	Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy
著者	Kitamura, Akira ; Ishida, Yoshihito ; Kubota, Hiroshi ; Pack, Chan-Gi ; Homma, Takayuki ; Ito, Shinya ; Araki, Kazutaka ; Kinjo, Masataka ; Nagata, Kazuhiro
e-Rad_Researcher 10580152 著者名 Kitamura, Akira
著者名 Ishida, Yoshihito
e-Rad_Researcher 80332724 著者名 Kubota, Hiroshi
著者名 Pack, Chan-Gi
著者名 Homma, Takayuki
著者名 Ito, Shinya
著者名 Araki, Kazutaka
e-Rad_Researcher 70177971 著者名 Kinjo, Masataka
e-Rad_Researcher 50127114 著者名 Nagata, Kazuhiro
言語	英語
キーワード	Molecular chaperone
	HSP47
	Collagen
	Fluorescence correlation spectroscopy
発行日	2018-02-10
出版者	Elsevier
収録物名	Biochemical and Biophysical Research Communications
巻(号)	497(1)
ページ	279-284
抄録	Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis.
資源タイプ	学術雑誌論文
出版タイプ	Accepted Manuscript
権利情報	Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
権利情報	©2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/
PISSN	0006-291X
関連情報	PMID 29438711
関連情報 (isVersionOf)	出版社版DOI https://doi.org/10.1016/j.bbrc.2018.02.069