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.

SUMMARYWe generated a knockout mouse for the neuronalspecific β-tubulin isoform Tubb3 to investigate its role in nervous system formation and maintenance. Tubb3−/− mice have no detectable neurobehavioral or neuropathological deficits, and upregulation of mRNA and protein of the remaining β-tubulin isotypes results in equivalent total b-tubulin levels in Tubb3−/− and wild-type mice. Despite similar levels of total β-tubulin, adult dorsal root ganglia lacking TUBB3 have decreased growth cone microtubule dynamics and a decreased neurite outgrowth rate of 22% in vitro and in vivo. The effect of the 22% slower growth rate is exacerbated for sensory recovery, where fibers must reinnervate the full volume of the skin to recover touch function. Overall, these data reveal that, while TUBB3 is not required for formation of the nervous system, it has a specific role in the rate of peripheral axon regeneration that cannot be replaced by other β-tubulins.

Summary 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-coils demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the C-terminal 7-heptad repeat dimer 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.

OBJECTIVES To develop a translational mouse model for the study and measurement of non-evoked pain in the orofacial region by establishing markers of nociceptive-specific grooming behaviors in the mouse. BACKGROUND Some of the most prevalent and debilitating conditions involve pain in the trigeminal distribution. Although there are current therapies for these pain conditions, for many patients they are far from optimal. Understanding the pathophysiology of pain disorders arising from structures innervated by the trigeminal nerve is still limited and most animal behavioral models focus on the measurement of evoked pain. In patients, spontaneous (non-evoked) pain responses provide a more accurate representation of the pain experience than do responses that are evoked by an artificial stimulus. Therefore, the development of animal models that measure spontaneous nociceptive behaviors may provide a significant translational tool for a better understanding of pain neurobiology. METHODS C57BL/6 mice received either an injection of 0.9% Saline solution or complete Freund’s adjuvant (CFA) into the right masseter muscle. Animals were video recorded and then analyzed by an observer blind to the experiment group. The duration of different facial grooming patterns performed in the area of injection were measured. After 2 hrs, mice were euthanized, perfused and the brainstem was removed. Fos protein expression in the trigeminal nucleus caudalis was quantified using immunohistochemistry to investigate nociceptive-specific neuronal activation. A separate group of animals was treated with morphine sulfate, to determine the nociceptive-specific nature of their behaviors. RESULTS We characterized and quantified 3 distinct patterns of acute grooming behaviors: fore-paw rubbing, lower lip skin/cheek rubbing against enclosure floor and hind paw scratching. These behaviors occurred with a reproducible frequency and time course, and were inhibited by the analgesic morphine. CFA-injected animals also showed Fos labeling consistent with neuronal activation in nociceptive-specific pathways of the trigeminal nucleus after two hours. CONCLUSIONS These behaviors and their correlated cellular responses represent a model of trigeminal pain that can be used to better understand basic mechanisms of orofacial pain and identify new therapeutic approaches to this common and challenging condition.

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.