Ubiquitination of Niemann-Pick Cl protein
| Project/Area Number |
18590295
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Tottori University |
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Principal Investigator |
NINOMIYA Haruaki Tottori University, Faculty of Medicine, Professor (80212124)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Niemann-Pick / cholesterol / ubiquitin / roteasome |
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| Research Abstract |
We and others reported reduced protein levels of NPC1 in fibroblasts from patients with NPC1 mutations. Importantly, cells from patients with juvenile/adult forms retained relatively high levels of the protein, suggesting that the NPC1 protein level may be one of the factors that determine the disease severity. We found that a proteasome inhibitor MG132 caused an accumulation of ubiquitinated NPC1, suggesting proteasomal degradation of NPC1s. Pulse-chase analysis in COS cells revealed that an I1061T mutation decreased the half-life time of expressed NPC1 protein. Wild-type as well as loss-of-function mutant NPC1 proteins associated with molecular chaperones HSP70, HSP90 and calnexin. Accordingly, overexpression of HSP70 in human fibroblasts with I1061T homozygous mutations by adeno-HSP70 or treatment with geranylgeranylacetone increased the level of the mutant protein. In COS cells, co-expression of an E3 ligase CHIP (carboxyl terminus of HSP70-interacting protein) enhanced MG132-induced accumulation of ubiquitinated NPC1. MALDI-TOF mass spectrometry has revealed three lysine residues on the cytosolic side, K318, K792 and K1180, as potential acceptors of ubiquitin. Substitution of the three lysine residues with alanine yielded a mutant protein with a steady-state level approximately 10 times higher than the wild-type protein. These findings indicate that NPC1 undergoes proteasomal degradation and that its biosynthesis is an inefficient process: 90% of the wild-type protein is degraded away. These findings also suggest the possibility to restore endosomal cholesterol flow by stabilization of the mutant NPC1 proteins.
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Report
(3 results)
Research Products
(19 results)
