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G.S. Katselis, G.S. Wu, R.E. Moore, N.A. Rao, T.D. Lee; Interaction of Cytochrome C With A–Crystallin in the Early Phase of Experimental Autoimmune Uveitis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4571.
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Nitrated cytochrome C released into the photoreceptor cytosol, during the late amplification phase of experimental autoimmune uveitis (EAU), is believed to be the precursor of procaspase 9 that initiates apoptosis thus resulting in retina damage. However, the early phase of EAU is not characterized by apoptosis which suggests that certain proteins interact with cytochrome C and prevent it from activating the caspases. Previous work indicated that up–regulated protein αA–crystallin may form a complex with cytochrome C and may in turn affect apoptosis. The goal of the present work is to fully characterize the interaction of αA–crystallin with cytochrome C in the early phase of EAU particularly with respect to the role of posttranslational modifications and the identification of other proteins that may be part of the complex.
EAU was induced in Lewis rats by injecting 50 µg of S–antigen in complete Freund’s adjuvant. A group of rats serving as the control were injected with the adjuvant alone. The rats were euthanized at day 5 following immunization, their retinas were removed and proteins isolated using procedures previously described in Wu et al. (2005). Immobilized antibodies to αA–crystallin and cytochrome C were used to isolate protein complexes. The gel separated proteins were reduced, alkylated, and digested in gel using published procedures. Tryptic peptides were extracted and analyzed by LC–MS using an Eksigent NanoLC–2D HPLC coupled to a LTQ–FT hybrid ion–trap/ICR mass spectrometer. Tandem mass spectra were searched against the NCBI non–redundant database using TurboSequest. Search results were evaluated using Qscore, a probability based scoring algorithm.
Preliminary results confirm the presence of the previously observed cytochrome C – αA–crystallin interactions. The possible role of other proteins identified in the complex will be discussed.
Detection of αA–crystallin by LC–MS and its association with cytochrome C, in the early phase of EAU, is a first indication that this protein plays a significant role in preventing apoptosis and thus retinal damage.
G. S. Wu, T. D. Lee, R. E. Moore and N. A. Rao (2005). Photoreceptor Mitochondrial Tyrosine Nitration in Experimental Uveitis. Investigative Ophthalmology and Visual Science, 46 (7), 2271 – 2281.
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