Rebecca K. Donegan

Structural basis for misfolding in myocilin-associated glaucoma.

Olfactomedin (OLF) domain-containing proteins play roles in fundamental cellular processes and have been implicated in disorders ranging from glaucoma, cancers and inflammatory bowel disorder, to attention deficit disorder and childhood obesity. We solved crystal structures of the OLF domain of myocilin (myoc-OLF), the best studied such domain to date. Mutations in myoc-OLF are causative in the autosomal dominant inherited form of the prevalent ocular disorder glaucoma. The structures reveal a new addition to the small family of five-bladed β-propellers. Propellers are most well known for their ability to act as hubs for protein-protein interactions, a function that seems most likely for myoc-OLF, but they can also act as enzymes. A calcium ion, sodium ion and glycerol molecule were identified within a central hydrophilic cavity that is accessible via movements of surface loop residues. By mapping familial glaucoma-associated lesions onto the myoc-OLF structure, three regions sensitive to aggregation have been identified, with direct applicability to differentiating between neutral and disease-causing non-synonymous mutations documented in the human population worldwide. Evolutionary analysis mapped onto the myoc-OLF structure reveals conserved and divergent regions for possible overlapping and distinctive functional protein-protein or protein-ligand interactions across the broader OLF domain family. While deciphering the specific normal biological functions, ligands and binding partners for OLF domains will likely continue to be a challenging long-term experimental pursuit, atomic detail structural knowledge of myoc-OLF is a valuable guide for understanding the implications of glaucoma-associated mutations and will help focus future studies of this biomedically important domain family.

The Glaucoma-associated Olfactomedin Domain of Myocilin Is a Novel Calcium Binding Protein

Background: Myocilin is an extracellular protein linked to glaucoma but is of unknown structure and function. Results: The myocilin olfactomedin domain contains a buried calcium ion ligated by Asp-380. Conclusion: The myocilin olfactomedin domain binds calcium with an unprecedented ligand arrangement. Significance: The presence of calcium within the OLF domain provides new clues into normal myocilin function, myocilin glaucoma pathogenesis, and biomedically important olfactomedin domains. Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure. Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF). Our study reveals an unprecedented high affinity calcium binding site within myoc-OLF. The calcium ion remains bound to wild-type OLF at neutral and acidic pH. A glaucoma-causing OLF variant, myoc-OLF(D380A), is calcium-depleted. Key differences in secondary and tertiary structure between myoc-OLF(D380A) and wild-type myoc-OLF, as well as limited access to chelators, indicate that the calcium binding site is largely buried in the interior of the protein. Analysis of six conserved aspartate or glutamate residues and an additional 18 disease-causing variants revealed two other candidate residues that may be involved in calcium coordination. Our finding expands our knowledge of calcium binding in extracellular matrix proteins; provides new clues into domain structure, function, and pathogenesis for myocilin; and offers insights into highly conserved, biomedically relevant OLF domains.

Discovery of Molecular Therapeutics for Glaucoma: Challenges, Successes, and Promising Directions.

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.

The Glaucoma-Associated Olfactomedin Domain of Myocilin Forms Polymorphic Fibrils That Are Constrained by Partial Unfolding and Peptide Sequence

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.

Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies.

Olfactomedin (OLF) domains are found within extracellular, multidomain proteins in numerous tissues of multicellular organisms. Even though these proteins have been implicated in human disorders ranging from cancers to attention deficit disorder to glaucoma, little is known about their structure(s) and function(s). Here we biophysically, biochemically, and structurally characterize OLF domains from H. sapiens olfactomedin-1 (npoh-OLF, also called noelin, pancortin, OLFM1, and hOlfA), and M. musculus gliomedin (glio-OLF, also called collomin, collmin, and CRG-L2), and compare them with available structures of myocilin (myoc-OLF) recently reported by us and R. norvegicus glio-OLF and M. musculus latrophilin-3 (lat3-OLF) by others. Although the five-bladed β-propeller architecture remains unchanged, numerous physicochemical characteristics differ among these OLF domains. First, npoh-OLF and glio-OLF exhibit prominent, yet distinct, positive surface charges and copurify with polynucleotides. Second, whereas npoh-OLF and myoc-OLF exhibit thermal stabilities typical of human proteins near 55°C, and most myoc-OLF variants are destabilized and highly prone to aggregation, glio-OLF is nearly 20°C more stable and significantly more resistant to chemical denaturation. Phylogenetically, glio-OLF is most similar to primitive OLFs, and structurally, glio-OLF is missing distinguishing features seen in OLFs such as the disulfide bond formed by N- and C- terminal cysteines, the sequestered Ca2+ ion within the propeller central hydrophilic cavity, and a key loop-stabilizing cation-π interaction on the top face of npoh-OLF and myoc-OLF. While deciphering the explicit biological functions, ligands, and binding partners for OLF domains will likely continue to be a challenging long-term experimental pursuit, we used structural insights gained here to generate a new antibody selective for myoc-OLF over npoh-OLF and glio-OLF as a first step in overcoming the impasse in detailed functional characterization of these biomedically important protein domains.

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin

Glaucoma-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.

Handling heme: The mechanisms underlying the movement of heme within and between cells

ABSTRACT Heme is an essential cofactor and signaling molecule required for virtually all aerobic life. However, excess heme is cytotoxic. Therefore, heme must be safely transported and trafficked from the site of synthesis in the mitochondria or uptake at the cell surface, to hemoproteins in most subcellular compartments. While heme synthesis and degradation are relatively well characterized, little is known about how heme is trafficked and transported throughout the cell. Herein, we review eukaryotic heme transport, trafficking, and mobilization, with a focus on factors that regulate bioavailable heme. We also highlight the role of gasotransmitters and small molecules in heme mobilization and bioavailability, and heme trafficking at the host‐pathogen interface. Graphical abstract Figure. No Caption available. HighlightsHeme is transported and trafficked within and between cells for use in hemoproteins and for signaling.The mechanisms underlying heme mobilization, trafficking, and transport, which are not well understood, are reviewed.A number of signaling molecules, including H2O2 and NO, regulate heme dynamics, bioavailability and trafficking.The transport and trafficking of heme in most human pathogens is required for virulence.