Abstract
Abstract: :
Purpose: We have previously found that in the early phase of experimental autoimmune uveitis (EAU), three major tyrosine (Tyr)–nitrated proteins in the retina were all mitochondria–related, with the most intensely nitrated protein being cytochrome c (cyto c). In this study, we further investigated the release and site of nitration of cyto c. In EAU, the initial disruption of mitochondrial homeostasis leading to nitric oxide synthase upregulation and protein nitration has not been reported. We looked for these early events in microarray analysis. Methods: Experimental autoimmune uveitis was induced in Lewis rats by bovine S–antigen. Affymetrix screening of 8800 rat genes was carried out using eight eyes each for both experimental and control runs. Mitochondria were isolated from the retinas and the release of cyto c was detected by UV/VIS absorption, Western blot, and liquid chromatography/tandem mass spectrometry (LC/MS/MS). These analyses were also compared with in vitro nitrated cyto c using 3–morphosydnonimine as a peroxynitrite donor. The release of cyto c was quantified through the course of EAU, on post–immunization days 0, 5, 10, 12, and 14. Site of nitration was determined by LC/MS/MS. Results: The substantial release of cyto c was seen in days 5 and 10; there was then a gradual decrease starting day 12, day 14 being the peak of inflammation. In cyto c nitrated in vitro, the preferential nitration site was Tyr–48 among four Tyr residues in the cyto c. The nitration of a critical heme–vicinal site, such as Tyr–48 is known to promote protein conformational change. In microarray analysis, 2– to 3–fold changes were seen in genes regulating cyto c oxidase subunits, calcium and ion channels, ATPase, fatty acid transporter (to mitochondria), and respiratory enzymes. In apoptosis–related genes, caspase–3 was down–regulated and serine protease inhibitor was upregulated. Conclusions: Genomic analysis suggests that in the early phase of EAU, there exists a subtle disturbance in mitochondrial homeostasis, resulting in opening of permeability transition pore and decrease in functions of respiratory chain. These changes could lead to the generation of peroxynitrite in mitochondria. Together with nitration and release of cyto c, these events in the early phase of EAU mark the initiation of inflammatory processes.
Keywords: mitochondria • gene microarray • nitric oxide