The myocilin olfactomedin domain (myoc-OLF) is linked to inherited forms of open angle glaucoma. Mutant myocilin accumulates within the endoplasmic reticulum of human trabecular meshwork cells leading to cell death and the build up of intraocular pressure, a common risk factor for glaucoma. In this work, a novel high affinity calcium-binding site buried within myoc-OLF was characterized. Additionally amyloidogenic peptide stretches within myoc-OLF that may be responsible for mutant myocilin aggregation were determined. Additionally the crystal structure of myoc-OLF was solved providing the first crystal structure of an olfactomedin domain protein. Insights from the structure into the relationship between disease causing mutations and myoc-OLF misfolding and the currently unknown function of myoc-OLF as explored by structural based prediction of ligand binding are also discussed.

Glaucoma is a neurodegenerative protein misfolding disorder classified by increases in IOP, damage to retinal ganglion cells (RGCs), optic nerve (ON) head damage, and progressive irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form of glaucoma, constituting over 90% of clinical cases. POAG is observed in patients where normal outflow channels, mainly the trabecular meshwork (TM), are exposed at the angle formed by the iris and cornea. However, due to TM cellular dysfunction, aqueous outflow resistance is increased preventing normal circulation of aqueous humor. Recent studies have shown that in 2-4% of POAG cases, increased intracellular levels of a secreted glycoprotein, called myocilin, are present in the TM. Myocilin is a 504aa glycoprotein, with an unknown precise function. Recent studies have postulated the importance of myocilin in oligodendrocyte differentiation, axonal myelination, and early apoptosis of retinal cells in development, but exact function is still widely debated. However, it is important to note that pathology associated with myocilin is only seen during POAG. Also, only cells of the TM exhibit toxicity when overexpressing mutant myocilin. In the normal eye, myocilin is secreted from the ER of TM cells, however, mutations in the MYOC gene lead to an aggregation-prone form of the protein, which is inefficiently processed and degraded from the ER, leading to build-up and associated toxicity. There are over 70 known MYOC mutations associated with glaucoma, with over 90% occurring in the C-terminal OLF domain. Some of the more common, pathological mutations are: I477N, Y437H, P370L, W286R, N480K. All of these mutations have been observed in patients with glaucoma, and all lead to build-up and aggregation of the mutant protein within the ER of TM cells. Recently, work out of our lab has discovered an interaction between mutant forms of myocilin and the chaperone Grp94. Grp94 is the resident Hsp90 isoform of the ER. Grp94 is an important chaperone in ER quality control, aiding in the output of properly folded secretory and membrane-bound proteins. Besides protein folding, other roles of Grp94 in the ER include: calcium buffering, roles in ER quality control (including targeting misfolded proteins for ERAD), peptide binding, and roles in ER stress. Generally, terminally misfolded proteins in the ER are degraded through ERAD; the Grp94 mediated shuttling of misfolded proteins to the ER trans-membrane machinery for ubiquitination and subsequent translocation to the cytosol for proteasomal degradation. However, in the case of POAG, ERAD is inefficient in mutant myocilin degradation, causing protein accumulation within the ER. In this study, we demonstrate that specific Grp94 inhibition of interaction with mutant or misfolded myocilin leads to myocilin degradation and subsequent lowering of protein accumulation in the TM, thus reducing downfield pathology associated with POAG. Grp94 preserves mutant myocilin in the ER of TM cells leading to protein accumulation and aggregation precipitating TM cellular dysfunction. We showed in various in vitro cell assays that Grp94 inhibition leads to a reduction in intracellular protein levels, while alleviating TM cellular toxicity. Furthermore, in the Tg-MYOCY437H mouse model of POAG, we showed that topical ocular administration of a specific Grp94 inhibitor alleviated glaucomatous pathologies, including elevated IOP, myocilin accumulation in the TM, reduced scotopic/photopic visual responses, and RGC health and viability. Finally, we have proven the importance of ER-stress pathway malfunction in the development of POAG pathology, while also discovering the involvement of the autophagy mechanism for myocilin degradation following Grp94 inhibition. Overall, this work proves that Grp94 is an important regulator of myocilin pathology during POAG. While there are no current therapeutics on the market that directly target the underlying POAG disease mechanism, specific Grp94 inhibition shows great promise and should be considered for human clinical trials. If successful, specific targeted Grp94 inhibition could be the first curative therapeutic options for patients suffering from myocilin-associated POAG.

Myocilin, expressed in the trabecular meshwork of the eye, has been linked to inherited primary open-angle glaucoma (POAG). The biological function of myocilin is unknown, but mutant myocilin exhibits a gain-of-function mechanism, aggregating within the endoplasmic reticulum of human trabecular meshwork cells, causing cell stress and eventually apoptosis. After apoptosis occurs, the trabecular meshwork is compromised, leading to an increase in intraocular pressure, a symptom of glaucoma. In this thesis, I have expressed and purified the wild-type olfactomedin (OLF) domain and 24 reported disease-causing variants. I developed a facile thermal stability assay using differential scanning fluorimetry, which follows the unfolding of a protein through the fluorescence of a dye sensitive to hydrophobic regions of a protein. Also in this thesis I have determined melting temperatures for the wild-type and for each of the disease-causing mutants. I have tested the stability of the mutants in the presence of seven osmolytes, with sarcosine and trimethylamine-N-oxide restoring the melting temperature closest to wild-type. Additionally, I expressed and purified three reported single nucleotide polymorphisms (SNPs) (E352Q, E396D, K398R), which are considered benign variants. Variants were also compared by circular dichroism, revealing high b-sheet content and wild-type structure. When compared to previous studies, there is a positive correlation between the melting temperature, and previously reported qualitative assays, which measure the mutant myocilin solubility in detergent, secretion from mammalian cells, and aggregation propensity. Taken together, these data give insight into the relationship between glaucoma genotypes and phenotypes.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.