Creation of a New Method to Refold the Membrane Protein Mutants for Curing of Hereditary Hearing Loss

创建一种膜蛋白突变体重折叠治疗遗传性听力损失的新方法

• 批准号: 20390439
• 负责人: WADA Hiroshi
• 金额: $ 12.23万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2008
• 资助国家: 日本
• 起止时间: 2008 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20390439/
• 关键词: 耳科学; 遺伝性難聴; ペンドリン; タンパク質フォールディング; Pendrin

The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the conditioning of the ion concentration in the endolymph of the inner ear. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. In this study, therefore, cellular localization and anion exchanger activity were analyzed by using HEK293 cells transfected with mutant genes. First, 10 pendrin mutants reported in Japanese patients were constructed. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that normal pendrin was transported to the plasma membrane ; however, 8 mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restored the function. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants from the cytoplasm to the plasma membrane and recovered the anion exchanger activity.

SLC26A4基因编码跨膜蛋白pendrin，其参与内耳内淋巴中离子浓度的调节。SLC26A4基因突变导致感音神经性听力损失。然而，造成这种损失的机制仍然不明。因此，在本研究中，通过使用转染有突变基因的HEK293细胞来分析细胞定位和阴离子交换活性。首先，构建了日本患者中报道的10种pendrin突变体。pendrin突变体的细胞定位的免疫荧光染色显示，正常pendrin被运送到质膜，然而，8个突变体被保留在细胞质中。此外，我们分析了水杨酸，作为一种药理伴侣，是否恢复了功能。用10 mM水杨酸盐孵育转染突变体的细胞，诱导4种pendrin突变体从细胞质转运到质膜，并恢复阴离子交换活性。

项目成果

Site-directed mutagenesis of the motor protein prestin

运动蛋白 prestin 的定点诱变

• 发表时间: 2010
• 作者: 内田 浩;荒瀬 透;小野政徳;各務真紀;小田英之;西川明花;丸山哲夫;吉村泰典;和田仁
• 通讯作者: 和田仁

Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin

• DOI: 10.1299/jbse.3.221
• 发表时间: 2008
• 期刊: Journal of Biomechanical Science and Engineering
• 作者: Koji Iida;M. Murakoshi;S. Kumano;K. Tsumoto;K. Ikeda;Toshimitsu Kobayashi;I. Kumagai;H. Wada

内耳モータータンパク質プレスチンの膜貫通構造解析-原子間力顕微鏡を用いて-(Analysis of transmembrane structure of the inner ear motor protein prestin-atomic force microscopy study-)

内耳运动蛋白prestin跨膜结构分析-原子力显微镜研究-

• 发表时间: 2011
• 作者: Iida K;Murakoshi M;Kumano S;Tsumoto K;Ikeda K;Kobayashi T;Kumagai I;Wada H;村越道生
• 通讯作者: 村越道生

人工脂質二鹸と原子間力騨鏡を利用しなタンパク質モータprestinの可視化

使用人工脂质和原子力镜对蛋白质运动 prestin 进行可视化

• 发表时间: 2010
• 作者: Ozawa N;Maruyama T;Nagashima T;Ono M;Arase T;Ishimoto H;Yoshimura Y;和田仁
• 通讯作者: 和田仁

High resolution imaging by atomic force microscopy of prestin purified and reconstituted into an artificial lipid bilayer

通过原子力显微镜对 prestin 进行高分辨率成像，纯化并重构为人工脂质双层

• 发表时间: 2010
• 作者: 浅田弘法;古谷正敬;西尾 浩;升田博隆;内田 浩;丸山哲夫;梶谷 宇;木挽貢慈;吉村泰典;鈴木翔;丸山哲夫;熊野峻
• 通讯作者: 熊野峻