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Hani Levkovitch-Verbin, Mike Fainzilber, Shely Vander, Juan A Oses-Prieto, Katalin F Medzihradszky, Alma Burlingame, Marek Rajman; Phosphoproteomics of Axonal Signaling in glaucoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2972.
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© ARVO (1962-2015); The Authors (2016-present)
To identify retrograde axonal signaling mechanisms activated in glaucoma using phosphproteomics.
Experimental glaucoma was induced unilaterally in 120 Wistar rats using the translimbal photocoagulation laser model. Optic nerves and retinas were removed separately from control and IOP-elevated eyes for phosphoproteomic characterization 5 days after the induction of glaucoma. Protein samples were reduced, alkylated and digested by trypsin. To quantify differences in the phosphorylation levels between the samples peptides were labeled with ITRAQ and enriched for phosphopeptides.. Labeled phosphopeptides were fractionated by strong cation exchange chromatography and analyzed by mass spectrometry. Transpath database (implemented in ExPlain) was used to construct signaling networks supported by the differentially phosphorylated protein in retina and optic nerve.
We have identified 38 and 60 differentially phosphorylated proteins in glaucomatous retinas and optic nerves, respectively. Glaucoma induced increase in the phosphorylation levels of more than 2/3 of differentially phosphorylated proteins (30 in retina, 49 in optic nerve), eight of them were identified both in retinas and in optic nerves (STAT3, Erk1, Erk2, Hsp27, Nestin, Palladin, Protein DEK and Protein Ag2).Bioinformatics analysis identified 4 molecules (Stat3, Erk1, Erk2 and PKCdelta) that are involved in common signaling pathways. In glaucoma the increase in the phosphorylation levels in the retinas and optic nerves were 2.6 and 2.2 fold for Erk1, 2.32 and 2.03fold for Erk2, 3.34 and 2.49 fold for STAT3 respectively, as compared to control samples. GO annotation analysis (DAVID bioinformatics) revealed that most of the differentially phosphorylated proteins are involved in regulation of cytoskeleton (microtubule, actin, and neurofilament cytoskeleton), cellular transport and phosphorylation.
Using quantitative phosphoproteomic approach we were able to identify almost 100 differentially phosphorylated proteins in glaucomatous retinas and optic nerves. Bioinformatics analysis suggests that phosphorylated STAT3, Erk1, Erk2 and PKCdelta are important molecules involved in common signaling pathways affected by glaucoma.
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