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N.A. Rao, G. Wu, T.D. Lee; Nitration of Mitochondrial Proteins Leads to Photoreceptor Degeneration . Invest. Ophthalmol. Vis. Sci. 2004;45(13):763.
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Purpose: We previously found three major tyrosine (Tyr) nitrated proteins in experimental uveitis (EAU) at the peak of inflammation. These three proteins, mitochondria import stimulation factor (MSF), phosphoglycerate mutase (PGM) and cytochrome c (cyto c), are all mitochondria–related proteins. Herein, we determined the site of nitration and release of cyto c in the course of EAU. Methods: EAU was induced in Lewis rats by bovine S–antigen. Nine rats each were killed on day(D)5, D10, D12, and D14 postimmunization, and 6 retinas were combined for one determination. Nine nonimmunized rats were used as controls (D0). The proteins were analyzed mainly by Western blots probed with anti–nitroTyr and anti–rat cyto c antibodies. MSF and PGM were identified by mass spectrometry. Retinal cytosol and intact mitochondria were separated by gradient centrifugation with 280 mM sucrose buffer. Mitochondrial outer membranes were disrupted by sonication to determine the release of cyto c into the intermembrane space, reflecting the extent of cyto c dissociated from the repiratory chain complex. Leukocyte infiltration in the retina was detected by histologic examination. Results:The intensities of nitrated mitochondria proteins were at maximum at D5 and D10 and leveled off after D10. At D5 and D10, the release of cyto c into the cytosol was not observed with the intact mitochondria. When mitochondrial outer membranes were disrupted, however, an abundant release of cyto c was seen in cytosol in D5 and D10, but not in D0 eyes. Conclusions: The present study revealed the nitration of mitochondrial proteins and subsequent loss of cyto c from the respiratory chain complex. Two simultaneous impairments are required for the release of cyto c to occur: disengagement of nitrated cyto c from the respiratory chain complex and disruption of mitochondrial outer membranes. These events were found to occur early (D5) in EAU, prior to any leukocyte infiltration. A major cause of nitration could be photoreceptor mitochondrial generation of peroxynitrite. Nitration of these proteins also resulted in loss of photoreceptor mitochondrial functions before macrophage infiltration.
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