Defining a link between gap junction communication, proteolysis, and cataract formation

Disruption of the connexin alpha3 (Cx46) gene (alpha3 (-/-)) in mice result s in severe cataracts within the nuclear portion of the lens. These catarac ts 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 catarac t formation and gamma -crystallin cleavage in alpha3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identifie d the calcium-dependent proteases, m-calpain and Lp82, as the primary targe ts of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased i n alpha3 (-/-) lenses and correlated both spatially and temporally with cat aract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alph a3 (-/-) lenses. These data establish a role for alpha3 gap junctions in ma intaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key ini tiator of the process of cataractogenesis.