Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones.
Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones.
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DOI：
10.1021/cb900282e
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发表时间：
2010-05-21
影响因子：
4
通讯作者：
Lieberman, Raquel L.
中科院分区：
文献类型：
--
作者：
Burns, J. Nicole;Orwig, Susan D.;Harris, Julia L.;Watkins, J. Derrick;Vollrath, Douglas;Lieberman, Raquel L.
肌球蛋白的突变导致遗传性开角型青光眼，这是一种与眼内压升高相关的普遍神经退行性疾病。肌球蛋白形成小梁网细胞外基质的一部分，推测其调节眼内压。错义突变，聚集在嗅调节蛋白（OLF）结构域的myocilin，使蛋白质易于聚集在内质网的小梁网细胞，引起细胞功能障碍和死亡。细胞研究表明，温度敏感的分泌的myocilin突变体，但在表达和纯化的困难，排除了生物物理特性的野生型（wt）myocilin和致病突变体在体外。我们已经通过纯化野生型和选择的引起大肠杆菌肉瘤的突变体（D380 A、I477 N、I477 S、K423 E）形式的OLF结构域（228-504）克服了这些限制，所述OLF结构域（228-504）与来自大肠杆菌的麦芽糖结合蛋白（MBP）融合。杆菌单体融合蛋白可以在溶液中分离。为了确定野生型和突变型OLF结构域的相对稳定性，我们开发了不去除MBP的荧光热稳定性测定，并提供了突变型OLF折叠但热稳定性低于野生型的第一个直接证据。我们测试了7种化学伴侣稳定突变型肌细胞素的能力。只有肌氨酸和三甲胺N-氧化物能够将所有测试的突变体的解链温度转移到接近wt OLF的解链温度。我们的工作为鉴定能够稳定突变myocilin并促进分泌到细胞外基质的定制小分子奠定了基础，以更好地控制眼内压并最终延迟myocilin青光眼的发作。
Mutations in myocilin cause an inherited form of open angle glaucoma, a prevalent neurodegenerative disorder associated with increased intraocular pressure. Myocilin forms part of the trabecular meshwork extracellular matrix presumed to regulate intraocular pressure. Missense mutations, clustered in the olfactomedin (OLF) domain of myocilin, render the protein prone to aggregation in the endoplasmic reticulum of trabecular meshwork cells, causing cell dysfunction and death. Cellular studies have demonstrated temperature-sensitive secretion of myocilin mutants, but difficulties in expression and purification have precluded biophysical characterization of wild-type (wt) myocilin and disease-causing mutants in vitro. We have overcome these limitations by purifying wt and select glaucoma-causing mutant (D380A, I477N, I477S, K423E) forms of the OLF domain (228–504) fused to maltose binding protein (MBP) from E. coli. Monomeric fusion proteins can be isolated in solution. To determine the relative stability of wt and mutant OLF domains, we developed a fluorescence thermal stability assay without removal of MBP, and provide the first direct evidence that mutated OLF is folded but less thermally stable than wt. We tested the ability of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and ultimately delay the onset of myocilin glaucoma.
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影响因子：
2.3
作者：
Goldwich, Andreas;Scholz, Michael;Tamm, Ernst R.
通讯作者：
Tamm, Ernst R.
影响因子：
4.4
作者：
Jia, Li-Yun;Gong, Bo;Yam, Gary Hin-Fai
通讯作者：
Yam, Gary Hin-Fai
影响因子：
4
作者：
Kennan, AM;Mansergh, FC;Farrar, GJ
通讯作者：
Farrar, GJ
影响因子：
2.9
作者：
Lieberman, Raquel L.;D'aquino, J. Alejandro;Ringe, Dagmar;Petsko, Gregory A.
通讯作者：
Petsko, Gregory A.
影响因子：
3.5
作者：
DIGUAN, C;LI, P;INOUYE, H
通讯作者：
INOUYE, H