Light-induced changes in protein nitration in photoreceptor rodouter segments

[摘要] Purpose: Light has been shown to modulate protein nitration in ratretinas. To better understand the role of protein nitration inphotoreceptor cell death induced by intense light, we examined retinalprotein nitration and identified target proteins in rod outer segments(ROS).Methods: Cyclic light-reared rats, treated or not with theantioxidant, dimethylthiourea (DMTU), were exposed to intense greenlight for 8 h. A subset of these rats was kept in the dark for 24 hafter 8 h of light exposure. Western analysis of ROS proteins with ananti-nitrotyrosine antibody was performed to examine changes in proteinnitration. 2D-immunoblots with anti-nitrotyrosine antibody followed byliquid chromatography tandem mass spectrometry was used to identifynitrated proteins in ROS. The expression levels of three nitric oxidesynthase (NOS) isoforms, inducible, neuronal-, and endothelial-NOS weresemi-quantified by immunoblot analysis.Results: Western analysis revealed that the level of ROS proteinnitration increased during the dark recovery period after 8 h of lighttreatment in both DMTU treated and untreated rats. However, DMTUeffectively reduced protein nitration in ROS during light exposure andduring the subsequent dark recovery period. Using 2D-immunoblottingfollowed by liquid chromatography tandem mass spectrometry analysis, weidentified ten ROS proteins as nitration targets. Most of these proteinswere glycolytic enzymes. The level of inducible-NOS in the retina wasincreased by light exposure.Conclusions: The effect of DMTU in reducing ROS protein nitrationduring and after light suggests the involvement of protein nitrationduring light-induced photoreceptor cell death. Nitration of glycolyticenzymes specifically may alter their activities. Increased levels ofiNOS during and after intense light exposure suggest that this isoformis responsible for intense light induced protein nitration in ROS duringthe dark recovery period. The limited nitration seen in ROS during lightexposure may reflect a quenching effect by endogenous antioxidants onthe generation of reactive oxygen and nitrogen species.

[学科分类] 生物化学/生物物理