Niemann-Pick type C1I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding
Niemann-Pick type C1I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding
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DOI：
10.1074/jbc.m708735200
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发表时间：
2008-03-28
影响因子：
4.8
通讯作者：
Ory, Daniel S.
中科院分区：
文献类型：
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作者：
Gelsthorpe, Mark E.;Baumann, Nikola;Ory, Daniel S.
在NPC1基因中已经发现了200多种致病突变。最普遍的突变是NPC1(I1061T)，预计位于富含半胱氨酸的腔结构域内，并与典型的少年发病型尼曼-皮克C型病相关。为了深入了解NPC1（I1061T）突变导致疾病的分子机制，我们检测了NPC1（I1061T）突变纯合子的人成纤维细胞中突变蛋白的表达。尽管野生型和NPC1（I1061）成纤维细胞之间的NPC1 mRNA水平相似，但NPC1蛋白水平在NPC1(I1061)T细胞中下降了85%。代谢标记研究表明，与野生型蛋白不同，NPC1（I1061T）蛋白经历了从远藤氢敏感到远藤氢抗性的糖基化模式转变，NPC1（I1061T）蛋白几乎完全是远藤氢敏感的，其半衰期（t(1/2) 6.5 h）比野生型远藤氢抗性（t(1/2) 42 h）缩短。使用化学伴侣、在允许温度下生长或抑制蛋白酶体降解会增加NPC1（I1061T）蛋白水平，这表明突变蛋白可能是内质网相关降解（ERAD）的靶标，原因是蛋白质错误折叠。NPC1缺陷细胞中NPC1（I1061T）的过表达导致突变蛋白的晚内体定位和NPC突变表型的互补，可能是由于一小部分新生的NPC1（I1061T）蛋白能够正确折叠并逃避内质网质量控制检查点。我们的发现首次描述了内质网运输缺陷作为人类鼻咽癌疾病的机制，揭示了NPC1（I1061T）突变导致疾病的机制，并提出了治疗由NPC1（I1061T）突变引起的鼻咽癌疾病的新方法。
Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1(I1061T), is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1(I1061T) mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1(I1061T) mutation. Despite similar NPC1 mRNA levels between wild type and NPC1(I1061T) fibroblasts, NPC1 protein levels are decreased by 85% in NPC1(I1061)T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1(I1061T) protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t(1/2) 6.5 h) versus wild type Endo H-resistant species (t(1/2) 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1(I1061T) protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1(I1061T) in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1(I1061T) protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1(I1061T) mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1(I1061T) mutation.