Mar 26, 2010

Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones

ACS Chemical Biology
J Nicole BurnsRaquel L Lieberman

Abstract

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 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 ...Continue Reading

  • References48
  • Citations27

References

  • References48
  • Citations27

Citations

Mentioned in this Paper

Alkalescens-Dispar Group
Methylamines
Aggregation
Extracellular Matrix
Trimethyloxamine
Mutant Proteins
Pancortin 4 protein, mouse
Eye Proteins
Process of Secretion
Olfactomedin

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