Abstract
Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.
Highlights
Gap junctions are formed by two hexameric structures of connexin molecules that interact with connexons in neighboring cells to form membrane aqueous pores [1]
Each cell is coupled to its neighbors via gap junctions, resulting in a network of cell-cell contacts that has been suggested to be important for the maintenance of ion flux and for metabolic cooperation between the peripheral lens cells and the interior fiber cells [4]
Since ␥-crystallin cleavage was previously reported to be associated with ␣3 (Ϫ/Ϫ) cataractogenesis [7], the processing of ␥-crystallin was analyzed during the onset of cataracts (Fig. 1B)
Summary
Connexin; AAH, artificial aqueous humor; NLVS, 5-iodo-4-hydroxy-3-nitrophenyl-acetyl-leucinyl-leucinylleucine vinyl sulfone; PAGE, polyacrylamide gel electrophoresis; WT, wild type; TBST, Tris-buffered saline with Tween 20; Z-VAD, Z-ValAla-Asp(OMe)-fluoromethyl ketone. New biochemical reagents have been generated that allow the monitoring of global changes in protease activity. These reagents take advantage of the broad reactivity of the natural product E-64 to create chemical probes that covalently react with the papain family of cysteine proteases in an activitydependent manner [20]. We propose that calcium accumulation and the subsequent activation of the lens-specific calpain Lp82 in the ␣3 (Ϫ/Ϫ) lens are key events leading to cataract formation. These studies provide a functional link between ␣3 gap junctions and maintenance of calcium homeostasis in the lens
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