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Alexandra M. Dins, Christopher J. Lieven, Leonard A. Levin; Cystatin-Related Protein is a Candidate Signaling Molecule for Cell Death After Experimental Retinal Ischemia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6057.
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Retinal ischemia followed by reperfusion causes production of reactive oxygen species (ROS) that may induce apoptosis. To elucidate the pathways underlying cell death after ischemia-reperfusion injury, we used a redox proteomic approach to identify molecules undergoing formation of intermolecular disulfide bonds.
Long Evans rats were anesthetized, and IOP was raised to 110 mmHg or maintained at 15 mm Hg (sham) in one eye for 60 min. The unoperated contralateral eye served as control. Retinas were removed immediately following ischemia or after 4 hrs of reperfusion and prepared for gel electrophoresis. Following a first dimension run in non-reducing conditions, excised gel lanes were incubated in dithiothreitol to reduce disulfide bonds, and then in iodoacetamide to alkylate free sulfhydryls. After incubations the gel lanes were run in the second dimension and stained with SYPRO Ruby. Spots that differed between conditions were picked and identified by matrix-assisted laser desorption/ionization/time-of-flight (MALDI/TOF) mass spectrometry.
The vast majority of proteins were on the 45 degree diagonal, indicating that they did not contain intermolecular disulfide bonds. Multiple spots were below the diagonal, indicating that they had reducible disulfide bonds when isolated from the retina, but these were present in all conditions (control, sham, ischemia, ischemia-reperfusion). A single spot was observed below the diagonal in multiple 4 hr reperfusion gels, which showed an increased intensity compared to the naïve, sham, and no reperfusion conditions, indicating the presence of ischemia/reperfusion-dependent formation of intermolecular disulfide bonds. Analysis by MALDI/TOF identified cystatin-related protein 1 precursor (CRP-1).
We identified CRP-1 as a protein which undergoes dimerization in ischemia-reperfusion. CRP-1 may be a redox-sensitive signaling molecule for retinal neuronal death in ischemia.
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