Rescue of Glaucoma-Causing Mutant Myocilin Thermal Stability by Chemical Chaperones

被引:49
作者
Burns, J. Nicole [1 ]
Orwig, Susan D. [1 ]
Harris, Julia L. [1 ]
Watkins, J. Derrick [1 ]
Vollrath, Douglas [2 ,3 ]
Lieberman, Raquel L. [1 ]
机构
[1] Georgia Inst Technol, Sch Chem & Biochem, Atlanta, GA 30332 USA
[2] Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA
[3] Stanford Univ, Sch Med, Dept Ophthalmol, Stanford, CA 94305 USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
MALTOSE-BINDING PROTEIN; OLFACTOMEDIN DOMAIN; RETICULUM STRESS; IN-VITRO; EXPRESSION; OSMOLYTE; JUVENILE; GENE; STABILIZATION; PURIFICATION;
D O I
10.1021/cb900282e
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, 1477N,14775, K423E) forms of the OLF domain (228-504) fused to a 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 to ultimately delay the onset of myocilin glaucoma.
引用
收藏
页码:477 / 487
页数:11
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