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G. Tezel, X. Yang, C. Luo, J. Cai; Proteomic Identification of 14-3-3 Protein Interactions in a Chronic Pressure-Induced Rat Model of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3283.
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In order to identify specific signaling molecules involved in glaucomatous neurodegeneration, proteomic analysis was performed to identify phosphorylated proteins and their interactions with other proteins following experimental elevation of intraocular pressure in rats.
Intraocular pressure elevation was induced in rats by hypertonic saline injections into episcleral veins. For retinal phosphoprotein enrichment, spin columns containing immobilized metal-affinity chromatography media were utilized, and enriched samples were subjected to gel-free protein identification using tandem mass spectrometry (LC-MS/MS). Since the initially identified phosphorylated proteins in ocular hypertensive retinas included the 14-3-3 family proteins, 14-3-3 was selected as target for the following experiments. To detect proteins interacting with 14-3-3, the 14-3-3-containing protein complexes were eluted using co-immunoprecipitation and recombinant protein-based affinity pull-down and subjected to LC-MS/MS analysis for protein identification. SDS-PAGE was performed to confirm the purified protein complexes. In addition, for further validation of the proteomic findings, western blot analysis and immunohistochemistry were performed using specific antibodies, which included phosphorylation site-specific antibodies.
Consecutive experiments through LC-MS/MS analysis of eluted protein complexes identified various proteins with significant sequence matches for multiple peptides. The identified proteins differentially interacting with 14-3-3 in ocular hypertensive versus normotensive control eyes included zinc-finger protein, calmodulin, protein kinase C, ubiquitin, heat shock protein 70, and a proapoptotic member of the Bcl-2 family, Bad. Western blots confirmed the identified proteins, and immunolabeling of retina sections with specific antibodies demonstrated their differential sub-cellular localizations in retinal ganglion cells.
Findings of this in vivo study through proteomic analysis identify that an important protein family associated with checkpoint control pathways, 14-3-3, is involved in cellular signaling during glaucomatous neurodegeneration in a phosphorylation-dependent manner. The 14-3-3 appears to constitute an important regulatory pathway of cell death signaling, which controls the subcellular localization, stabilization, and function of many important proteins associated with retinal ganglion cell death.
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