Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations
Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations
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DOI：
10.1111/j.1471-4159.2007.04457.x
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发表时间：
2007-06
影响因子：
4.7
通讯作者：
Norimasa Takahashi;M. Morita;T. Maeda;Y. Harayama;N. Shimozawa;Yasuyuki Suzuki;H. Furuya;R. Sato;Y. Kashiwayama;T. Imanaka
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作者：
Norimasa Takahashi;M. Morita;T. Maeda;Y. Harayama;N. Shimozawa;Yasuyuki Suzuki;H. Furuya;R. Sato;Y. Kashiwayama;T. Imanaka
X染色体肾上腺脑白质营养不良基因（ALD; ABCD 1）突变导致X连锁肾上腺脑白质营养不良（X-ALD），这是一种严重的神经退行性疾病。编码的肾上腺脑白质营养不良蛋白（ALDP/ABCD 1）是人类中745个氨基酸的一半大小的过氧化物酶体ATP结合盒蛋白。在这项研究中，我们选择了9种具有天然错义突变的任意突变型人ALDP形式（R104 C，G116 R，Y174 C，S342 P，Q544 R，S606 P，S606 L，R617 H和H667 D），并检查了细胞内行为。当在缺乏ALDP的X-ALD成纤维细胞中表达时，突变型His-ALDP（S606 L、R617 H和H667 D）的表达水平低于野生型和其他突变型ALDP的表达水平。此外，在CHO细胞中稳定表达的突变ALDP-绿色荧光蛋白（S606 L和H667 D）由于快速降解而未检测到。有趣的是，在这些细胞中共表达的野生型ALDP也消失了。在来自ALD患者（R617 H）的X-ALD成纤维细胞的情况下，在细胞中未检测到突变ALDP，但在与蛋白酶体抑制剂孵育后出现。当表达突变ALDP-绿色荧光蛋白（H667 D）的CHO细胞在蛋白酶体抑制剂存在下培养时，突变型和野生型ALDP均重新出现。此外，通过免疫荧光研究，在跨膜结构域2和3之间具有突变的突变体His-ALDP（Y174 C）未表现出过氧化物酶体定位。这些结果表明，在ALDP的COOH末端一半具有突变的突变ALDP，包括S606 L，R617 H和H667 D，在二聚化后被蛋白酶体降解。此外，跨膜结构域2和3之间的区域对于ALDP靶向过氧化物酶体是重要的。
Mutation in the X‐chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X‐linked adrenoleukodystrophy (X‐ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half‐size peroxisomal ATP‐binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X‐ALD fibroblasts lacking ALDP, the expression level of mutant His‐ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP‐green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co‐expressed in these cells also disappeared. In the case of X‐ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP‐green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His‐ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH‐terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.