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

肌浆网Ca^2+-ATP酶（SERCA）是肌浆网Ca^2+的转运酶，参与ATP的水解，在维持Ca^2+稳态中发挥重要作用。Ca^2+转运位点位于跨膜区，而催化位点位于胞质的三个区域（A、P和N）。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（E1 PCa_2），当Ca^<2+>释放到管腔中并水解后，该EP异构化为ADP不敏感型（E2 P）。在EP异构化过程中，三个胞质结构域的相对位置和相互作用方式发生了很大变化。这些结构变化被传递到跨膜结构域以诱导Ca^<2+>释放。作者发现，连接A结构域和M1的环（A/M1接头）的长度对于从EP形成到EP水解发生的事件是重要的。最重要的是，它对Ca^< 关于我们 2+>从EP释放到管腔中。结果表明，EP异构化反应是由两个连续的步骤组成的，而以前认为是一个单一的步骤。首次发现并捕获了介于E1PCa_2和E2 P之间的中间态E2PCa_2.作者研究了在Darier病（DD）家系中发现的与肌浆网Ca^<2+>-ATP酶2b（SERCA 2b）51个突变相关的可能缺陷。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，但即使是轻微的稳态紊乱也会导致疾病。我们的研究结果也为深入了解SERCA的结构与功能关系提供了新的思路，并揭示了该酶的关键区域和残基. ATP 2 Cl是位于高尔基体的Ca^<2+>/Mn^<2+>-ATP酶，被认为是Hailey-Hailey病的致病基因。但其在表皮中的定位和作用尚未完全阐明。作者发现ATP 2Cl特异性定位于正常表皮的基底细胞层。无论是角质形成细胞从培养皿中分离，也不治疗与高浓度的钙抑制ATP 2C 1的表达，而这两个程序诱导分化标志物，K10角蛋白和外皮蛋白。相反，ATP 2C 1的敲低诱导培养的角质形成细胞的这些分化标志物。结果表明，ATP 2 Cl起着至关重要的作用，为基底角质形成细胞保持在未分化状态，其减少引起分化和向上定位到基底上层最有可能通过锰饥饿在高尔基体的角质形成细胞。少

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