A New Luminescence-Based Assay for Monitoring Mutant Myocilin Cellular Localization
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Harris, Ethan Foley
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Abstract
Glaucoma is a heterogenous eye disease that is the leading cause of irreversible blindness worldwide, caused by a blockage in the outflow of aqueous humor from the eye, leading to an increase in intraocular pressure. The most common glaucoma subtype is primary open angle glaucoma, affecting ~60 million people worldwide. 2-4% of primary open angle glaucoma (POAG) and 10% of juvenile open angle glaucoma (JOAG) cases are attributed to mutations in the gene encoding for the protein myocilin. One of the key characteristics of a pathogenic variant is its inability to be secreted from trabecular meshwork cells. Instead of secretion, pathogenic myocilin variants accumulate in the endoplasmic reticulum and causes cell death.
Mutant myocilins are studied in the lab using cellular secretion assays. In the past, different cellular fractions were detected using immunoblot, which has sensitivity limitations. In this thesis, we implement a new method that utilizes HiBiT, a luciferase-based assay, which we hypothesized would be more sensitive than immunoblot.
The myocilin mutants tested using HiBiT (T377K, T377R, D384G, D395ins, C433Y, T455K, and L486F) to detect myocilin cellular localization exhibited negligible secretion when compared to WT. In addition, all the mutants except for L486F accumulated intracellularly. Unexpectedly, L486F was not detected intracellularly to the same degree as the other mutants, as evidenced by low band intensity in both the western and HiBiT blots for all three fractions.
HiBiT blotting provides more sensitive detection than traditional immunoblotting, as evidenced by increased band intensity in the HiBiT blots for the same sample and lower quantities of cells needed for robust detection.
Further studies on L486F will be required to determine whether it is poorly expressed or being degraded intracellularly. Overall, HiBiT detection of mutant myocilin enables new insights to be made into the mechanism of myocilin-associated POAG.
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2024-07-30
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