Characterization of purified myocilin: glaucoma as a protein misfolding disease

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DESCRIPTION (provided by applicant): Glaucoma, the leading cause of blindness in the world, has no direct treatment. Eye drops, laser treatment, and/or surgery medically manage the symptom of increased intraocular pressure observed prior to retinal degeneration and loss of visual field, but do not constitute a therapy. Pressure is controlled in the anterior region of the eye, within the anatomical pathway for outflow of aqueous humor, called the trabecular extracellular meshwork (TEM). Of the ~70 million glaucoma cases, ~4% comprise an autosomal-dominant, inherited form closely linked to mutations in myocilin, a TEM component. Over 90% of the mutations in myocilin occur within its olfactomedin (OLF) domain, which is highly conserved among higher eukaryotes but is of unknown structure and function. Glaucoma-causing mutant myocilins are poorly secreted out of human trabecular meshwork (HTM) cells to the TEM. Instead, they accumulate in the endoplasmic reticulum (ER), resulting in abnormal TEM morphology, HTM cell death, and early-onset glaucoma. Interestingly, homozygous and heterozygous myocilin knock-out mice, and individuals with myocilin truncation mutations, exhibit no ocular abnormalities. Thus, pathogenesis is likely caused by a toxic gain-of-function mechanism, placing a significant number of glaucoma cases within the framework of a disease of protein misfolding and mistrafficking. The primary objective of this proposal is to characterize purified wild-type and mutant myocilins using solution biophysical techniques and X-ray crystallography to gain a better understanding of myocilin structure, function, and folding. Although difficulties in recombinant expression have limited in vitro characterization of myocilin thus far, we have recently filled this need for the key OLF domain. We have purified monomers, cytosolic aggregates, and insoluble inclusions from our recombinant system. The proposed work will lead to a molecular understanding of the pathogenesis in inherited and age-onset glaucoma, and thereby broaden our knowledge of protein conformational disorders and the function of OLF domains in biology. The proposed studies will also provide new opportunities for drug discovery for myocilin glaucoma, both "bottom-up" via a new stability assay, as well as "top-down" using the three dimensional structure. To date, our in vitro studies support the hypothesis that mutant myocilins retain near wild-type structure but exhibit compromised stability. We further hypothesize that these defects are recognized by ER quality control machinery, which prevents the mutant myocilins from being secreted to the TEM. The accumulation of mutant myocilin provokes cell stress responses that lead to HTM cell death. Thus, our long-term goal is to use the knowledge of myocilin structure, stability, and folding to identify small molecules that enable mutant myocilin to escape detection by ER quality control and prevent aggregation in the TEM. This treatment would reduce ER accumulation and pathogenic downstream events, and thereby directly delay the onset of intraocular pressure and subsequent vision loss for inherited glaucoma patients. PUBLIC HEALTH RELEVANCE: Our long-term goal is to develop a new therapy for glaucoma, a prevalent eye disease characterized by increased intraocular pressure, neurodegeneration of the retina, and vision loss. We are studying myocilin, an extracellular matrix protein involved in regulating eye pressure; mutations in myocilin lead to early-onset, inherited forms of glaucoma. We will study myocilin and disease-causing mutants in terms of their structure and stability, which will guide our efforts to develop an assay that will identify therapeutic compounds that bind to, and stabilize, mutant myocilin to delay the onset of inherited glaucoma.