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Age-related cataract is a major cause of blindness worldwide, and cortical cataract is the second most prevalent type of age-related cataract. Although a significant fraction of age-related cataract is heritable, the genetic basis remains to be elucidated. We report that homozygous deletion of Epha2 in two independent strains of mice developed progressive cortical cataract. Retroillumination revealed development of cortical vacuoles at one month of age; visible cataract appeared around three months, which progressed to mature cataract by six months. EPHA2 protein expression in the lens is spatially and temporally regulated. It is low in anterior epithelial cells, upregulated as the cells enter differentiation at the equator, strongly expressed in the cortical fiber cells, but absent in the nuclei. Deletion of Epha2 caused a significant increase in the expression of HSP25 (murine homologue of human HSP27) before the onset of cataract. The overexpressed HSP25 was in an underphosphorylated form, indicating excessive cellular stress and protein misfolding. The orthologous human EPHA2 gene on chromosome 1p36 was tested in three independent worldwide Caucasian populations for allelic association with cortical cataract. Common variants in EPHA2 were found that showed significant association with cortical cataract, and rs6678616 was the most significant in meta-analyses. In addition, we sequenced exons of EPHA2 in linked families and identified a new missense mutation, Arg721Gln, in the protein kinase domain that significantly alters EPHA2 functions in cellular and biochemical assays. Thus, converging evidence from humans and mice suggests that EPHA2 is important in maintaining lens clarity with age.

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

Age-related cataract is a major cause of blindness worldwide, and cortical cataract is the second most prevalent type of age-related cataract. Although a significant fraction of age-related cataract is heritable, the genetic basis remains to be elucidated. We report that homozygous deletion of Epha2 in two independent strains of mice developed progressive cortical cataract. Retroillumination revealed development of cortical vacuoles at one month of age; visible cataract appeared around three months, which progressed to mature cataract by six months. EPHA2 protein expression in the lens is spatially and temporally regulated. It is low in anterior epithelial cells, upregulated as the cells enter differentiation at the equator, strongly expressed in the cortical fiber cells, but absent in the nuclei. Deletion of Epha2 caused a significant increase in the expression of HSP25 (murine homologue of human HSP27) before the onset of cataract. The overexpressed HSP25 was in an underphosphorylated form, indicating excessive cellular stress and protein misfolding. The orthologous human EPHA2 gene on chromosome 1p36 was tested in three independent worldwide Caucasian populations for allelic association with cortical cataract. Common variants in EPHA2 were found that showed significant association with cortical cataract, and rs6678616 was the most significant in meta-analyses. In addition, we sequenced exons of EPHA2 in linked families and identified a new missense mutation, Arg721Gln, in the protein kinase domain that significantly alters EPHA2 functions in cellular and biochemical assays. Thus, converging evidence from humans and mice suggests that EPHA2 is important in maintaining lens clarity with age.

As the starting point for folding, the properties of the denatured state ensemble are critical to the thermodynamics and kinetics of protein folding. We used methionine oxidation to destabilize monomeric λ repressor and predominantly populate the denatured state under non-denaturing buffer conditions. The denatured ensemble of lambda repressor comprises conformations that are compact. The hydrodynamic radius of the denatured state determined under physiological conditions by sedimentation velocity and pulsed-field gradient NMR is only slightly larger than the native state and much smaller than the highly-denatured state. These conformations are likely compact due to a significant degree of α-helical structure and some tertiary interactions indicated by circular dichroism. To determine which residues populate helical conformations, we used NMR spectroscopy. The chemical shift index (CSI) calculations for the Cα and Hα atoms indicate that the region of the protein corresponding to helix 1 in the native state has a significant α-helical population in the denatured state. Most of the short-range and medium-range assigned NOEs detected by NOESY spectroscopy are in this same region. There are NOEs in other regions of the protein, but they are not consistent with a highly helical conformations in any other region of the protein. No long-range NOEs were assigned. 15N NMR relaxation parameters were measured at two static magnetic fields to determine the backbone dynamics of the denatured state of monomeric lambda repressor. Reduced spectral density mapping and Lipari-Szabo model-free analysis were used to develop a molecular interpretation of the relaxation parameters. There are two regions of reduced conformational flexibility in this denatured state, with a region of flexibility between them. These two rigid regions correspond to protein sequence that consists of bulky amino acids that bury a large amount of surface area upon folding. Therefore, it is likely that steric hindrance leads to conformational restriction in unfolded proteins, not necessarily nascent secondary structure formation.