•Glaucoma-causing extracellular myocilin is associated with amyloid formation•Coiled-coil domain adopts a tripartite parallel dimer-of-dimers structure•Leucine zipper exhibits non-canonical heptad repeat pattern with disulfide cap•Glaucoma-associated variants in tetramer region alter structure but not stabilityGlaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.Download high-res image (201KB)Download full-size image

Glaucoma, a heterogeneous ocular disorder affecting ∼60 million people worldwide, is characterized by painless neurodegeneration of retinal ganglion cells (RGCs), resulting in irreversible vision loss. Available therapies, which decrease the common causal risk factor of elevated intraocular pressure, delay, but cannot prevent, RGC death and blindness. Notably, it is changes in the anterior segment of the eye, particularly in the drainage of aqueous humor fluid, which are believed to bring about changes in pressure. Thus, it is primarily this region whose properties are manipulated in current and emerging therapies for glaucoma. Here, we focus on the challenges associated with developing treatments, review the available experimental methods to evaluate the therapeutic potential of new drugs, describe the development and evaluation of emerging Rho-kinase inhibitors and adenosine receptor ligands that offer the potential to improve aqueous humor outflow and protect RGCs simultaneously, and present new targets and approaches on the horizon.

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min–1 × 10–3), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.

Mutations in the olfactomedin domain of myocilin (myoc-OLF) are the strongest link to inherited primary open angle glaucoma. In this recently identified protein misfolding disorder, aggregation-prone disease variants of myocilin hasten glaucoma-associated elevation of intraocular pressure, leading to vision loss. Despite its well-documented pathogenic role, myocilin remains a domain of unknown structure or function. Here we report the first small-molecule ligands that bind to the native state of myoc-OLF. To discover these molecules, we designed a general label-free, mix-and-measure, high throughput chemical assay for restabilization (CARS), which is likely readily adaptable to discover ligands for other proteins. Of the 14 hit molecules identified from screening myoc-OLF against the Sigma-Aldrich Library of Pharmacologically Active Compounds using CARS, surface plasmon resonance binding studies reveal three are stoichiometric ligand scaffolds with low micromolar affinity. Two compounds, GW5074 and apigenin, inhibit myoc-OLF amyloid formation in vitro. Structure–activity relationship-based soluble derivatives reduce aggregation in vitro as well as enhance secretion of full-length mutant myocilin in a cell culture model. Our compounds set the stage for a new chemical probe approach to clarify the biological function of wild-type myocilin and represent lead therapeutic compounds for diminishing intracellular sequestration of toxic mutant myocilin.

Here we present a multidisciplinary educational unit intended for general, advanced placement, or international baccalaureate-level high school science, focused on the three-dimensional structure of proteins and their connection to function and disease. The lessons are designed within the framework of the Next Generation Science Standards to make learning more relevant to daily life, and to help high school students engage in and understand advanced topics beyond the typical high school chemistry or biology curriculum. The unit involves lectures, videos, a hands-on activity, a research paper, a laboratory experiment, and a culminating project. Students are introduced to protein crystallography, the protein data bank, and the computer program PyMOL (free download for educational use) to visualize protein structure in three dimensions. Clear improvements in student comprehension of protein structure and function have been documented after implementation of the unit.Keywords: Amino Acids; Biochemistry; Computer-Based Learning; Enzymes; Hands-On Learning/Manipulatives; High School/Introductory Chemistry; Inquiry-Based/Discovery Learning; Molecular Modeling; Proteins/Peptides; X-ray Crystallography;

Myocilin variants, localized to the olfactomedin (OLF) domain, are linked to early-onset, inherited forms of open-angle glaucoma. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix of the eye. We hypothesize that, like in other diseases of protein misfolding, aggregation and downstream pathogenesis originate from the compromised thermal stability of mutant myocilins. In an expansion of our pilot study of four mutants, we compare 21 additional purified OLF variants by using a fluorescence stability assay and investigate the secondary structure of the most stable variants by circular dichroism. Variants with lower melting temperatures are correlated with earlier glaucoma diagnoses. The chemical chaperone trimethylamine N-oxide is capable of restoring the stability of most, but not all, variants to wild-type (WT) levels. Interestingly, three reported OLF disease variants, A427T, G246R, and A445V, exhibited properties indistinguishable from those of WT OLF, but an increased apparent aggregation propensity in vitro relative to that of WT OLF suggests that biophysical factors other than thermal stability, such as kinetics and unfolding pathways, may also be involved in myocilin glaucoma pathogenesis. Similarly, no changes from WT OLF stability and secondary structure were detected for three annotated single-nucleotide polymorphism variants. Our work provides the first quantitative demonstration of compromised stability among many identified OLF variants and places myocilin glaucoma in the context of other diseases of protein misfolding.

Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-β-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

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 of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and to ultimately delay the onset of myocilin glaucoma.

Human lysosomal enzymes acid-β-glucosidase (GCase) and acid-α-galactosidase (α-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and α-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using α-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of α-Gal A with DGJ. Both GCase and α-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase−IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in α-Gal A are not seen. Thermodynamic parameters obtained from α-Gal A unfolding indicate two-state, van’t Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and α-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.

•Aggregates of mutant myocilin are linked to early-onset, inherited glaucoma.•The myocilin olfactomedin domain forms amyloid fibrils via partially unfolded states.•Fibrils appear as straight/curvilinear and as a circular species of micron length.•Two peptide stretches fibrillize into these disparate morphologies.•Observed fibril morphologies may contribute to glaucoma pathogenesis.The glaucoma-associated olfactomedin domain of myocilin (myoc-OLF) is a recent addition to the growing list of disease-associated amyloidogenic proteins. Inherited, disease-causing myocilin variants aggregate intracellularly instead of being secreted to the trabecular meshwork, which is a scenario toxic to trabecular meshwork cells and leads to early onset of ocular hypertension, the major risk factor for glaucoma. Here we systematically structurally and biophysically dissected myoc-OLF to better understand its amyloidogenesis. Under mildly destabilizing conditions, wild-type myoc-OLF adopts non-native structures that readily fibrillize when incubated at a temperature just below the transition for tertiary unfolding. With buffers at physiological pH, two main endpoint fibril morphologies are observed: (a) straight fibrils common to many amyloids and (b) unique micron-length, ~ 300 nm or larger diameter, species that lasso oligomers, which also exhibit classical spectroscopic amyloid signatures. Three disease-causing variants investigated herein exhibit non-native tertiary structures under physiological conditions, leading to a variety of growth rates and a fibril morphologies. In particular, the well-documented D380A variant, which lacks calcium, forms large circular fibrils. Two amyloid-forming peptide stretches have been identified, one for each of the main fibril morphologies observed. Our study places myoc-OLF within the larger landscape of the amylome and provides insight into the diversity of myoc-OLF aggregation that plays a role in glaucoma pathogenesis.Download high-res image (113KB)Download full-size image

Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.Download high-res image (134KB)Download full-size imageResearch Highlights► Aggregates of mutant myocilin are closely associated with early-onset glaucoma. ► In vitro, myocilin aggregates exhibit the classical characteristics of amyloid. ► Amyloid has been localized to several regions of the OLF domain of myocilin. ► Full-length myocilin P370L variant accumulates amyloid in mammalian cell culture. ► Myocilin amyloids may contribute to the pathogenesis of inherited and sporadic glaucoma.