Molecular basis for regulation of intracellular environment and function of ion transporting proteins

调节细胞内环境和离子转运蛋白功能的分子基础

• 批准号: 17370046
• 负责人: KANAZAWA Hiroshi
• 金额: $ 8.83万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370046/
• 关键词: regulation of ion environment; membrane ion transport; Na^+; H^+ antiporters; calcineurin homologus protein 1; Bioenergetics ofmembrane transport; 膜イオン輸送機構; 膜輸送の分子機構; 蛋白質の機能解析; 細胞内の蛋白質局在化; 生体エネルギー共役; 蛋白質工学; 分子遺伝学

Intracellular concentrations of ions including pH and Na^+ are maintained at certain values. Na^+/H^+ antiporters required for these regulations exit in cytoplasmic and endocytic membranes as well and play a central role. In this study we aimed to clarify ion transport mechanisms including the binding sites of the trasporting ions in the antiporters. Main results of this study are classified into the following three groups. (1) The structure and function relationship of H.pylori Na^+/H^+ antiporter NhaA (HPNhaA) has been studied by means of biochemical and molecular biological approaches. The residues involved in the ion binding and transport have been newly identified for HPNhaA and the results were published in Biochemistry ( two other manuscripts are in preparation for publication). To determine the atomic structure of NhaA, a large scale of purified HPNhaA was successfully prepared to make a crystal. Further a new approach using FRET analysis has been successfully applied to detect … More conformational changes expected for ion transporting process (JBC (2005)). (2) Three new isoforms of human Na^+/H^+ antiporter (NHE) have been identified and revealed to localize in endocytic membranes. These isoforms were clarified to function as a K^+/H^+ antiporter and localize in different compartments depending on isoform type (JBC(2005)). DT40 cells with knocked out gene for CHP which can bind to NHE1 has been established. In these cells, CHP is missing and NHE1 activity was reduced to less than 90 % of the wild type. Surprisingly NHE was degraded in these cells, indicating that CHP is essentail for stabilizing NHE. This gives us a new concept of CHP1 with NHE (Amer.J.Phys (2007)). Further we determined crystal structure of CHP1. The crystal structure suggested a binding mechanism of CHP1 wuth NHE1 (JBC(2005)). (3) We have established a new procedure to detect Nhalp activity of S. cereviciae. Application of this method gave us stoichiometry of ions (Na^+/H^+) being electrogenic (BBA(2005)). The Nhalp was found to form a dimer which is essential in functioning (BBA(2005)).Our achievements were presented in several meeting including 2006 International Biochemistry Meeting, European Bioenergetics meeting, and so on. Less

包括 pH 和 Na^+ 在内的细胞内离子浓度维持在一定值，这些调节所需的 Na^+/H^+ 逆向转运蛋白也存在于细胞质和内吞膜中，并在本研究中发挥核心作用。阐明离子转运机制，包括反向转运蛋白中转运离子的结合位点。本研究的主要结果分为以下三组：（1）H.pylori Na^+/H^+的结构和功能关系。通过生化和分子生物学方法对反向转运蛋白 NhaA (HPNhaA) 进行了研究，新鉴定出 HPNhaA 涉及离子结合和转运的残基，结果发表在《生物化学》上（另外两篇手稿正在准备发表）。为了确定 NhaA 的原子结构，成功制备了大规模纯化的 HPNhaA 来制造晶体。此外，使用 FRET 分析的新方法已成功应用于检测预期的构象变化。离子转运过程 (JBC (2005))。 (2) 人类 Na^+/H^+ 反向转运蛋白 (NHE) 的三种新异构体已被鉴定并揭示定位于内吞膜中。 K^ +/H^+ 逆向转运蛋白并根据亚型类型定位在不同的区室中（JBC(2005) 具有敲除的 CHP 基因，可以结合）。在这些细胞中，CHP 缺失，NHE1 活性降低至野生型的 90% 以下。令人惊讶的是，NHE 在这些细胞中被降解，这表明 CHP 对于稳定 NHE 至关重要。 CHP1 与 NHE 的概念（Amer.J.Phys (2007)）进一步确定了 CHP1 的晶体结构。该晶体结构表明了 CHP1 的结合机制。 NHE1 (JBC(2005))。 (3) 我们建立了一种检测酿酒酵母 Nhalp 活性的新程序，应用该方法为我们提供了生电离子 (Na^+/H^+) 的化学计量（BBA(2005)）。 ))) 发现 Nhalp 形成了对功能至关重要的二聚体 (BBA(2005))。我们的成果在包括 2006 年国际会议在内的多次会议上进行了展示。生物化学会议、欧洲生物能量学会议等较少。

项目成果

Nuclear localization of the serine/threonine kinase DRAK2 is required for UV induced

丝氨酸/苏氨酸激酶 DRAK2 的核定位是 UV 诱导所需的

• 发表时间: 2006
• 期刊: Biol.Pharm.Bullt 280
• 作者: Miyazaki;E. et al.;Hiroshi Kuwahara 他
• 通讯作者: Hiroshi Kuwahara 他

Characterization of the ion transport activity of budding yeast Na^+/H^+ antiporter, Nha1p, on the secretory vesicles

芽殖酵母 Na^ /H^ 逆向转运蛋白 Nha1p 在分泌囊泡上的离子转运活性的表征

• 发表时间: 2005
• 期刊: Biochem.Biophys.Act Biomembrane 1712
• 作者: 土田 雅史;山下 ゆかり;小森 雅之;木田 祐一郎;阪口 雅郎;Ryuichi Ohgaki 他
• 通讯作者: Ryuichi Ohgaki 他

Characterization of the ion transport activity of budding yeast Na^+/H^+ antiporter, Nhalp, on the secretory vesicles

芽殖酵母 Na^ /H^ 逆向转运蛋白 Nhalp 在分泌囊泡上的离子转运活性的表征

• 发表时间: 2005
• 期刊: Biochim. Biophys. Act. 1712
• 作者: 土田 雅史;山下 ゆかり;小森 雅之;木田 祐一郎;阪口 雅郎;Ryuichi Ohgaki他
• 通讯作者: Ryuichi Ohgaki他

Structure-function relationship of the fifth transmembrane domain in the Near antiporer of Helicobacter pylori : Topology and function of the residues including two consecutive essential aspartate residues.

幽门螺杆菌近抗孔器第五跨膜结构域的结构-功能关系：包括两个连续必需天冬氨酸残基的残基的拓扑和功能。

• 发表时间: 2006
• 期刊: Biochemistry 45
• 作者: Naoyuki Kuwabara;Hiroki Inoue;Yumi;Tsuboi;Keiji Mitsui;Masafumi Matsushita;Hiroshi Kanazawa
• 通讯作者: Hiroshi Kanazawa

Structural characterization of calcineurin B homologous protein 1

• DOI: 10.1074/jbc.m503390200
• 发表时间: 2005-09-16
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Naoe, Y;Arita, K;Shimizu, T
• 通讯作者: Shimizu, T