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

Sarco(Endo)质网Ca~(2+)&t；~(2+)-ATPase(SERCA)将Ca~(2+)~(2+)离子从细胞质转运到管腔内，与ATP水解酶偶联，在Ca~(2+)内稳态中起着重要作用。Ca~(2+)~(2+)转运部位位于跨膜区，而催化部位位于A、P、N三个胞质区域。在钙离子转运循环中，泵首先被三磷酸腺苷磷酸化，产生磷酸化中间体(EP)。该内切酶为ADP敏感型(E1PCa_2)，释放Ca~(2+)进入管腔后异构化为ADP不敏感型(E2P)。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的变化。这些结构变化被传递到跨膜区，诱导钙释放。作者发现，连接A结构域和M1(A/M1连接子)的环的长度对于从EP形成到EP水解的事件是重要的。最重要的是，它对Ca^&lt；…至关重要更多的2+&>；从EP释放到管腔。结果表明，环氧乙烷异构化反应由连续的两步反应组成，以前认为是单步反应。首次发现并俘获了E1PCa_2和E2P之间的中间态E2PCa_2。作者检测了在Darier病(DD)家系中发现的SERCA2b(SERCA2b)与其51个突变相关的可能的SERCA2b缺陷。结果表明，在大多数情况下(51例中的48例)，DD突变通过蛋白表达和/或转运功能的缺陷而导致严重的钙稳态紊乱，从而导致DD，但即使是轻微的稳态紊乱也会导致疾病。我们的研究结果也进一步揭示了SERCA的结构与功能关系，揭示了该酶的关键区和残基。ATP2Cl是一种定位于高尔基体中的钙离子ATP酶，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，ATP2Cl在正常表皮中特异性地定位于基底细胞层。无论是角质形成细胞从培养皿中剥离还是高浓度钙处理都不能抑制ATP2C1的表达，而这两种方法都能诱导分化标记物K10角蛋白和总蛋白的表达。相反，ATP2C1基因的敲除诱导了培养的角质形成细胞的这些分化标志物。结果表明，ATP2Cl对基底层角质形成细胞维持未分化状态起着重要作用，其减少可能是通过角质形成细胞高尔基体中的锰饥饿诱导分化并向上定位到基底层。较少

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