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Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Published on: December 18, 2013
Temperature sensitive secretion of mutant myocilins
1Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA. vollrath@genome.stanford.edu
Abstract:
Recent studies have demonstrated that glaucoma-causing mutant myocilin proteins are misfolded and retained in the endoplasmic reticulum of cells. We showed previously that P370L mutant myocilin is poorly secreted at 37 degrees C and prolonged expression of the protein in differentiated human trabecular meshwork cells results in abnormal morphology and cell killing. Culturing cells at a lower temperature, a condition known to facilitate protein folding, enhances secretion and reverses the cytotoxic effects. We wanted to determine if temperature sensitive secretion is a general property of myocilin missense mutants. Wild-type or mutant forms of myocilin were transiently expressed in HEK 293 cells cultured at either 37 or 30 degrees C and protein secretion was assessed by immunoblotting. Of 15 myocilin missense mutants tested, representing a range in severity of associated glaucoma phenotypes, 14 displayed increased secretion at 30 degrees C. The sole exception was K423E, which is associated with an unusual mode of glaucoma inheritance. Generally, there is an inverse relationship between the degree of mutant myocilin secretion at 30 degrees C and the severity of the associated glaucoma phenotype. Mutants that show abundant secretion at 30 degrees C such as T377M, G364V, I499F and D380A are associated with less virulent glaucoma phenotypes, while mutants such as P370L, I477N, and Y437H display little secretion at 30 degrees C and are associated with more virulent glaucoma phenotypes. We conclude that temperature sensitive secretion is a property of most olfactomedin-domain myocilin mutants. The correlation between temperature sensitive secretion and glaucoma phenotype likely reflects the intrinsic susceptibility to misfolding of individual mutant proteins. These results support the hypothesis that myocilin-induced glaucoma is a protein conformational disease. Facilitating mutant protein folding could be a new approach to development of therapies for this disease.
Insights
Most glaucoma-causing myocilin mutants show temperature-sensitive secretion, with less severe glaucoma linked to better secretion. This suggests glaucoma is a protein misfolding disease, offering new therapeutic targets.
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Glaucoma is often caused by mutant myocilin proteins that misfold and accumulate in the endoplasmic reticulum.
- Previous work showed P370L mutant myocilin impairs trabecular meshwork cells, but lower temperatures improve its secretion and cell survival.
Purpose of the Study:
- To investigate if temperature-sensitive secretion is a common characteristic of various myocilin missense mutants.
- To correlate the degree of secretion at reduced temperatures with the severity of associated glaucoma phenotypes.
Main Methods:
- Transiently expressed wild-type and 15 myocilin missense mutants in HEK 293 cells.
- Cultured cells at either 37°C or 30°C to assess protein secretion via immunoblotting.
Main Results:
- 14 out of 15 myocilin mutants exhibited enhanced secretion at 30°C compared to 37°C.
- A general inverse correlation was observed between mutant myocilin secretion at 30°C and glaucoma phenotype severity.
- Mutants with high secretion at 30°C (e.g., T377M, G364V) are linked to milder glaucoma, while poorly secreted mutants (e.g., P370L, I477N) are linked to more severe forms.
Conclusions:
- Temperature-sensitive secretion is a characteristic of most olfactomedin-domain myocilin mutants.
- The findings support the hypothesis that myocilin-induced glaucoma is a protein conformational disease.
- Enhancing mutant protein folding presents a potential therapeutic strategy for glaucoma.
