Metrics & MoreArticle Recommendations CONSPECTUS: Numerous human disorders arise due to the inability of a particular protein to adopt its correct threedimensional structure in the context of the cell, leading to aggregation. A new addition to the list of such protein conformational disorders is the inherited subtype of glaucoma. Different and rare coding mutations in myocilin, found in families throughout the world, are causal for early onset ocular hypertension, a key glaucoma risk factor. Myocilin is expressed at high levels in the trabecular meshwork (TM) extracellular matrix. The TM is the anatomical region of the eye that regulates intraocular pressure, and its dysfunction is associated with most forms of glaucoma. Disease variants, distributed across the 30 kDa olfactomedin domain (mOLF), cause myocilin to be sequestered intracellularly instead of being secreted to the TM extracellular matrix. The working hypothesis is that the intracellular aggregates cause a toxic gain of function: TM cell death is thought to lead to TM matrix dysfunction, hastening elevated intraocular pressure and subsequent vision loss.Our lab has provided molecular underpinnings for myocilin structure and misfolding, placing myocilin-associated glaucoma within the context of amyloid diseases like Alzheimer and diabetes. We have dissected complexities of the modular wild-type (WT) myocilin structure and associated misfolded states. Our data support the model that full-length WT myocilin adopts a Y-shaped dimer-of-dimers conferred by two different coiled-coil regions, generating new hypotheses regarding its mysterious function. The mOLF β-propellers are paired at each tip of the Y. Disease-associated variants aggregate because mOLFs are less stable, leading to facile aggregation under physiological conditions (37 °C, pH 7.2). Mutant myocilin aggregates exhibit numerous characteristics of amyloid in vitro and in cells, and aggregation proceeds from a partially folded state accessed preferentially by disease variants at physiological conditions. Interestingly, destabilization is not a universal consequence of mutation. We identified counterintuitive, stabilizing point variants that adopt a non-native structure and do not aggregate; however, these variants have not been identified in glaucoma patients. An ongoing effort is predicting the consequence of any given mutation. This effort is relevant to interpreting data from large-scale sequencing projects where clinical and family history data are not available. Finally, our work suggests avenues to develop disease-modifying precision medicines for myocilin-associated glaucoma.

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Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

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