EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

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

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

<div><p>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.</p> </div

SPR detection of 3 binding to multiple caspase-6 surfaces confirms uncompetitive binding mode.

<p>(A) Catalytically inactive caspase-6 (green), apo-caspase-6 (blue) and caspase-6 saturated with VEID-FMK inhibitor (purple) were captured to chip surfaces and exposed to VEID-AMC, (VEID)₂R110 and/or <b>3</b> to qualitatively monitor binding. Cooperative binding of <b>3</b> and (VEID)₂R110 to C163 caspase-6 illustrate formation of the Michaelis-Menten complex. (B) Sensograms representing injections of escalating concentrations of <b>3</b> over VEID-FMK inhibitor-blocked caspase-6 surface (black). The inset represents similar injections of <b>3</b> over an unblocked apo-caspase-6 surface (blue). (C) Concentration-response analysis of data from (B) when compound <b>3</b> was injected over VEID-blocked caspase-6 surface (black) and apo-caspase-6 (blue) surfaces.</p

Structure of the <i>N</i>-furoyl-phenylalanine screening hit (2) and the optimized analog 3.

<p>Potency values represent the inhibition of caspase-6 cleavage of (VEID)₂R110 substrate.</p

Methionine oxidation of monomeric λ repressor: The denatured state ensemble under nondenaturing conditions

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric λ repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of α-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant

Docking models of caspase-6/VEID-R110/3 ternary complex explains fluorophore-dependent potency of this series of compounds.

<p>(A) Docking model of the Michaelis-Menten complex formed between caspase-6 (light blue), VEID-R110 (green sticks) and <b>3</b> (wheat sticks). (B) Docking model of the tetrahedral intermediate between caspase-6, VEID-R110 (green sticks) and <b>3</b> (wheat sticks) with substrate covalently bound to Cys163. (C) Depiction of monovalent VEID substrates with R110 or AMC fluorophores.</p

Kinetic caspase-6 enzymatic studies with compound 3 show uncompetitive mechanism of inhibition with (VEID)₂R110 substrate.

<p>(A) The initial enzyme velocity of caspase-6 was plotted against the indicated concentration of (VEID)₂R110 substrate in the presence of 0 nM (DMSO-black), 3 nM (red), 10 nM (orange), 30 nM (green) or 100 nM (blue) compound <b>3</b>. Double reciprocal plot of this data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s001" target="_blank">Figure S1</a> and Michaelis-Menten constants can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s006" target="_blank">Table S3</a>. (B) Concentration-response analysis of compound <b>3</b> when tested in the presence of 0.5 µM (red), 5 µM (black) or 20 µM (blue) (VEID)₂R110 substrate. Michaelis-Menten kinetic experiments were performed with single points while concentration-response curves were performed in duplicate. Each data set represents 1 of at least 3 experiments with similar results.</p

Inhibitor potency and selectivity against caspase family members.

<p>(A) Schematic of divalent tetrapeptide substrate proteolysis to release R110 fluorophore. Removal of both tetrapeptides by caspases is required for signal generation at 535 nm. Concentration-response analysis of compound <b>3</b> (B) and VEID-CHO (C) against caspase-6 (green), caspase-3 (black or red) or caspase-7 (blue). The particular divalent R110 peptide substrate used with each enzyme is indicated in the figure key and assay specifics can be found in Experimental Procedures. Potency values for (B–C) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s005" target="_blank">Table S2</a>. Concentration response curves were generated in duplicate and represent 1 of at least 2 experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. Data represent mean ± standard error of the mean.</p

Crystal structure of caspase-6 ternary complex with 3 and covalently bound VEID inhibitor reveals the uncompetitive mechanism of this series of compounds.

<p>(A) Crystal structure of the ternary complex of caspase-6 with zVEID and compound <b>3</b> (PDB-ID 4HVA). The caspase-6 dimer is represented as cartoon with the A and B chains colored light blue and grey, respectively, and the L4 loop colored purple. The zVEID inhibitors are represented as sticks and are colored pink. Each inhibitor is covalently bound to the catalytic cysteine (Cys163) in both chain A and B. Two molecules of <b>3</b> are shown as ball and stick representation and colored orange. (B) Close up of the active site of chain A colored according to (A) with hydrogen bonds shown as black dashes. (C) Structural comparison of caspase-6 ternary complex with <b>3</b> bound (light blue) and caspase-6 binary complex with bound VEID-CHO (wheat) (PDB-ID 3OD5) illustrating the difference in the conformation of the tip of the L4 loop in the two crystal structures (residues 261–271).</p