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，该蛋白参与调节内耳内淋巴中的离子浓度。 SLC26A4 基因突变会导致感音神经性听力损失。然而，造成这种损失的机制仍然未知。因此，在本研究中，通过使用转染突变基因的 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 个 pendrin 突变体。对pendrin突变体细胞定位的免疫荧光染色表明，正常的pendrin被转运至质膜；然而，8个突变体保留在细胞质中。此外，我们分析了水杨酸盐作为药理学伴侣是否恢复了功能。将突变体转染的细胞与 10 mM 水杨酸盐一起孵育，诱导 4 个 pendrin 突变体从细胞质转运至质膜，并恢复阴离子交换活性。

项目成果

Purification of the motor protein prestin from Chinese hamster ovary cells stably expressing prestin

从稳定表达prestin的中国仓鼠卵巢细胞中纯化运动蛋白prestin

• 发表时间: 2008
• 作者: Iida K; Murakoshi M; Kumano S; Tsumoto K; Ikeda K; Kobayashi T; Kumagai I; Wada H
• 通讯作者: Wada H

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: 和田仁

Topological characterization by atomic force microscopy of prestin in the plasma membrane of prestin-transfected chinese hamster ovary cells using quantum dots

使用量子点通过原子力显微镜对转染 prestin 的中国仓鼠卵巢细胞质膜中 prestin 进行拓扑表征

• 发表时间: 2008
• 作者: Murakoshi M; et.al.
• 通讯作者: et.al.

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Analysis of transmembrane structure of prestin by single molecule force spectroscopy

单分子力谱分析prestin的跨膜结构

• 发表时间: 2010
• 作者: 村越道生