Abstract
Purpose :
αB-crystallin has been identified to preserve lens cell viability upon UV-light and oxidative stress exposures, however, the lens organelle targets of αB-crystallin and its mechanisms for the protection of lens organelles have yet to be fully established. Here, we sought to establish the role and mechanisms for αB-crystallin protection of lens mitochondria and nuclei upon UV-light and oxidative stress exposures.
Methods :
Viability following exposure to UVA-light or H2O2-mediated oxidative stress was measured using the MTS assay. Mitochondrial function was measured using the membrane potential dependent dye JC-1 and mitochondrial ROS were measured using MitoSox fluorescent dye. Translocation of αB-crystallin was determined using immunocytochemistry and western blot analysis. Overexpression of chaperone mutant αB-crystallins was used to determine the role of chaperone function in mitochondrial protection.
Results :
αB crystallin prevented lens epithelial cell death upon exposure to UVA-light and oxidative stress insults. Strikingly, exposures to these insults resulted in specific phosphorylation of αB-crystallin and its translocation to lens mitochondria and nuclei. Purified αB directly interacted with cytochrome c (cyt c) to prevent its oxidation. Overexpression of αB prevented loss of mitochondrial membrane potential and release of cyt c in lens epithelial cells exposed to UVA and oxidative insult. Nuclear translocation of αB-crystallin resulted in the binding to nuclear speckles. Protection of mitochondrial function by αB-crystallin was independent of its well-established chaperone activity.
Conclusions :
The data provide evidence that lens cells respond to UV-light and oxidative stress insult through phosphorylation and translocation of αB to the mitochondria and nucleus. In the mitochondria αB prevents mitochondrial ROS production and cyt c release. In the nucleus, αB likely prevents against DNA damage. These novel organelle-specific functions for αB likely participate in its ability to prevent UV-light- and oxidative stress-mediated protein oxidation and apoptosis of lens cells to preserve lens transparency and prevent cataract formation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.