Conformatioanl Change in Phosphorylated Intermediate of Sarcoplasmic Reticulum Calcium Pump during Calcium Transport

肌浆网钙泵磷酸化中间体钙转运过程中的构象变化

• 批准号: 18570102
• 负责人: DAIHO Takashi
• 金额: $ 2.49万
• 依托单位: Asahikawa Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570102/
• 关键词: Ca^<2+>-ATPase; Ca^<2+>; M^<2+>-ATPase; sarco (endo) plasmic reticulum; Darier disease; keratinocyte; Golgi apparatus; calcium; conformation; sarco(endo)plasmic reticulum; site-directed mutagenesis; P-type ATPase; cation transport; phosphoryl

Sarco (endo) plasmic reticulum Ca^<2+>-ATPase (SERCA) transport Ca^<2+> ions from cytoplasm into lumen coupled with ATP hydrolysis, and has essential roles in Ca^<2+> homeostasis. The Ca^<2+> transport sites are located in transmembrane domain, while catalytic site is in the three cytoplasmic domains (A, P, and N). In the Ca^<2+> transport cycle, the pump is first phosphorylated by ATP to produce phosphorylated intermediate (EP). This EP is ADP-sensitive type (E1PCa_2) and isomerized to be ADP-insensitive type (E2P) after releasing Ca^<2+> into lumen and then hydrolyzed. The relative positions and interaction mode of the three cytoplasmic domains largely change in the EP isomerization. These structural changes are transmitted to the transmembrane domain to induce Ca^<2+> release.1. The author found that the length of the loop linking the A domain and Ml (A/M1 linker) is important for the events occurring from EP formation to EP hydrolysis. Above all, it is critically important for Ca^< … More 2+> release from EP into lumen. The results indicated that the EP isomerization, which had been thought to be a single step, is composed of consecutive two steps. The author identified and trapped the intermediate state E2PCa_2 between E1PCa_2 and E2P for the first time.2. The author examined possible defects of sarco (endo) plasmic reticulum Ca^<2+>-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier disease (DD) pedigrees. The results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca^<2+> homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.3. ATP2Cl is a Ca^<2+>/Mn^<2+>-ATPase localized in the Golgi apparatus and known as responsible gene for Hailey-Hailey disease. But its localization and roles in the epidermis are not fully elucidated. The author found that ATP2Cl is specifically localized at the basal cell layer in normal epidermis. Neither detachment of keratinocyte from culture dish nor treatment with high concentrations of calcium suppressed ATP2C1 expression, while both procedures induced differentiation markers, K10 keratin and involucrin. In contrast, knockdown of ATP2C1 induced these differentiation markers of cultured keratinocytes. The results suggest that ATP2Cl plays an essential role for basal keratinocytes to keep in the undifferentiated state and that its reduction evokes differentiation and up-localization to suprabasal layers most likely via the manganese starvation in the Golgi apparatus of keratinocytes. Less

肉质(内)质网Ca ^ 2+ -ATP酶(SERCA)将Ca ^ 2+ 离子从细胞质转运到管腔中并进行ATP水解，并且在Ca ^ 2+ 稳态中具有重要作用。 Ca 2+ 转运位点位于跨膜结构域中，而催化位点位于三个细胞质结构域(A、P和N)中。在Ca^2+转运循环中，泵首先被ATP磷酸化，产生磷酸化中间体(EP)。该EP为ADP敏感型(E1PCa_2)，向管腔释放Ca 2+ 后异构化为ADP不敏感型(E2P)，然后水解。 EP异构化过程中三个胞质结构域的相对位置和相互作用模式发生了很大的变化。这些结构变化被传递到跨膜结构域以诱导Ca^2+释放。1．作者发现连接A结构域和M1(A/M1接头)的环的长度对于从EP形成到EP水解发生的事件很重要。最重要的是，Ca^< … More 2+> 从 EP 释放到管腔中至关重要。结果表明，原本被认为是一步的EP异构化是由连续的两个步骤组成的。作者首次识别并捕获了E1PCa_2和E2P之间的中间状态E2PCa_2。 2．作者检查了肌(内)质网Ca^2+-ATP酶2b(SERCA2b)的可能缺陷，该缺陷与其在Darier病(DD)谱系中发现的51个突变相关。结果表明，在大多数情况下(51例中的48例)DD突变通过蛋白质表达和/或转运功能的缺陷导致Ca^2+稳态的严重破坏，从而导致DD，但即使稳态的轻微紊乱也会导致疾病。我们的结果还进一步深入了解了SERCA 的结构-功能关系，并揭示了该酶的关键区域和残基。3． ATP2Cl是位于高尔基体中的Ca ^ 2+ /Mn ^ 2+ -ATP酶并且已知为Hailey-Hailey病的负责基因。但其在表皮中的定位和作用尚未完全阐明。作者发现ATP2Cl特异性定位于正常表皮的基底细胞层。将角质形成细胞从培养皿中分离出来或用高浓度钙处理都不会抑制 ATP2C1 表达，而这两种方法都会诱导分化标记物 K10 角蛋白和外皮蛋白。相反，敲除 ATP2C1 会诱导培养的角质形成细胞的这些分化标记物。结果表明，ATP2Cl 对于基底角质形成细胞保持未分化状态起着至关重要的作用，并且其减少很可能通过角质形成细胞高尔基体中的锰饥饿引起分化并向上定位至基底上层。较少的

项目成果

筋小胞体カルシウムポンプ:リン酸化中間体の転換にともなうCa^<2+>の脱閉塞機構

肌浆网钙泵：与磷酸化中间体转化相关的 Ca^<2+> 解闭机制

• 发表时间: 2007
• 作者: Furuya;H;大保 貴嗣;大保 貴嗣
• 通讯作者: 大保 貴嗣

Role of the regions linking cytoplasmic and transmembrane domains in processing of phosphorylated intermediates for Ca^<2+> transport of sarcoplasmic reticulum Ca^<2+>-ATPase

连接细胞质和跨膜结构域的区域在肌浆网 Ca^2-ATPase 磷酸化中间体加工中的作用

• 发表时间: 2006
• 作者: Tatsuta;H.;Takashi Daiho
• 通讯作者: Takashi Daiho

筋小胞体Ca^<2+>-ATPaseのCa^<2+>放出過程におけるTry122-Hydrophobic clusterの役割

Try122-疏水簇在肌浆网Ca^2+-ATPase释放Ca^2+过程中的作用

• 发表时间: 2006
• 作者: Furuya;H.;山崎 和生
• 通讯作者: 山崎 和生

筋小胞体Ca^<2+>-ATPaseのP-Nヒンジ領域に存在する荷電側鎖の役割

带电侧链在肌浆网Ca^2+-ATP酶P-N铰链区的作用

• 发表时间: 2007
• 作者: Furuya;H;大保 貴嗣;大保 貴嗣;山崎 和生
• 通讯作者: 山崎 和生

筋小胞体Ca^<2+>-ATPaseの変異体によるCa^<2+>を閉塞したADP非感受性リン酸化中間体のアナログの形成

通过肌质网Ca 2+ -ATP酶突变体封闭Ca 2+ 的ADP不敏感磷酸化中间体类似物的形成

• 发表时间: 2007
• 作者: Furuya;H;大保 貴嗣
• 通讯作者: 大保 貴嗣