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

Sarco （endodo）质网Ca^<2+>-ATP酶（SERCA）通过ATP水解将Ca^<2+>离子从细胞质转运到腔内，在Ca^<2+>的稳态中起重要作用。Ca^<2+>转运位点位于跨膜结构域，而催化位点位于三个细胞质结构域（A、P和N）。在Ca^<2+>转运周期中，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），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，三个细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。这些结构变化传递到跨膜结构域，诱导Ca^<2+>释放。作者发现连接A结构域和Ml （A/M1连接体）的环的长度对于EP形成到EP水解的过程很重要。最重要的是，Ca^<…More 2+>从EP释放到管腔是至关重要的。结果表明，原认为是单一步骤的EP异构化实际上是由连续的两个步骤组成的。首次发现并捕获了介于E1PCa_2和E2P之间的中间态E2PCa_2。作者研究了sarco （endo）质网Ca^<2+>- atp酶2b （SERCA2b）可能与在Darier病（DD）家系中发现的51个突变相关的缺陷。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/或转运功能的缺陷导致Ca^<2+>内稳态严重破坏，从而导致DD，但即使是轻微的内稳态干扰也会导致疾病。我们的研究结果也进一步揭示了SERCAs的结构-功能关系，并揭示了该酶的关键区域和残基。ATP2Cl是一种Ca^<2+>/Mn^<2+>- atp酶，定位于高尔基体，被认为是黑利病的致病基因。但其在表皮中的定位和作用尚未完全阐明。笔者发现，ATP2Cl特异定位于正常表皮的基底细胞层。无论是从培养皿中剥离角质细胞，还是高浓度钙处理，都不能抑制ATP2C1的表达，但这两种方法都能诱导分化标志物K10角蛋白和天花素。相反，敲低ATP2C1可诱导培养的角质形成细胞的这些分化标记。结果表明，ATP2Cl在基底角化细胞保持未分化状态中起着至关重要的作用，其减少可能通过角化细胞高尔基体中的锰饥饿引起分化并向上定位到基底上层。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