Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder
Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder
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DOI：
10.1203/01.pdr.0000169984.89199.69
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发表时间：
2005-08-01
影响因子：
3.6
通讯作者：
Kondo, N
中科院分区：
文献类型：
--
作者：
Hashimoto, K;Kato, Z;Kondo, N
过氧化物酶体生物发生障碍包括齐薇格综合征和较轻的表型，如新生儿肾上腺脑白质营养不良（NALD）。我们先前对NALD患者的研究显示，PEX 13蛋白（Pex 13 p）的SH 3结构域中存在突变（Ile 326 Thr），显示过氧化物酶体生物发生中的温度敏感（TS）表型。临床TS表型在一些遗传性疾病中也有报道，但其分子机制仍有待阐明。用抗Pex 13 p抗体进行的免疫荧光染色也揭示了患者细胞中1326 T突变蛋白本身的TS表型。重组Pex 13 p-SH 3结构域的蛋白酶消化显示蛋白酶敏感性增加，表明突变蛋白折叠的问题。利用尿素梯度凝胶电泳或色氨酸残基的荧光发射对尿素变性的构象分析显示，突变蛋白应该容易展开。远紫外圆二色性（CD）光谱表明，野生型和突变体蛋白都有反平行的β-折叠作为其二级结构，但程度略有不同。热去折叠曲线显示1326 T蛋白的解链温度明显低于野生型蛋白。蛋白质的三维结构分析表明，Ile 326应该是一个核心残基的折叠动力学和Ile 326的苏氨酸取代应直接改变动力学平衡，这表明了显着增加的未折叠分子时，病人有高烧。对其他遗传性疾病中蛋白质的结构分析可以为更好地理解基因型-表型相关性提供途径。
Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the 1326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of protease susceptibility, suggesting a problem of mutant protein fold. Conformational analyses against urea denaturation using urea gradient gel electrophoresis or fluorescence emission from tryptophan residue revealed that the mutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as their secondary structure with slightly different extent. The thermal unfolding profiles measured by CD showed a marked lower melting temperature for 1326T protein compared with that of wild-type protein. Analysis of the protein 3D-structure indicated that the Ile326 should be a core residue for folding kinetics and the substitution of Ile326 by threonine should directly alter the kinetic equilibrium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlations.