| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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^<
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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
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