| Project/Area Number |
09470228
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Nagoya University |
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Principal Investigator |
SAITO Hidehiko School of Medicine, Nagoya University, Proffeser, 医学部, 教授 (20153819)
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| Co-Investigator(Kenkyū-buntansha) |
TOWATARI Masayuki School of Medicine, Medical Staff, 医学部, 医員
KOJIMA Tetsuhito School of Medicine, Assistant Proffeser, 医学部, 助手 (40161913)
TAKAMATSU Junnki School of Medicine, Associate Proffeser, 医学部, 助教授 (80221365)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥12,400,000 (Direct Cost: ¥12,400,000)
Fiscal Year 1998: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1997: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Keywords | Thrombophilia / protein C deficiency / gene mutation / impaired secretion / carboxyl-terminaldeletion / expression / conformational change / molecular model / プロテインS欠乏症 / nonsense mutation / 血小板mRNA / exon skipping / alternative splicing / AAV / プロテインS / 遺伝子治療 |
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| Research Abstract |
We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G) . This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381 -P419) by 81 abnormal amino acids. This elongated mutation was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible foe the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401 -P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degrated intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.
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