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Research

Targets of Tyrosine Nitration in Diabetic Rat Retina^*,S

Xianquan Zhan^,, Yunpeng Du^¶, John S. Crabb^,, Xiaorong Gu^,, Timothy S. Kern^¶,||,**, and John W. Crabb^,,,¶¶,||||

From the Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, Departments of ^¶ Medicine, ^|| Ophthalmology, Chemistry, and ^** Center for Diabetes Research, Case Western Reserve University, Cleveland, Ohio 44106, Research Service, Cleveland Veterans Affairs Medical Center, Cleveland Ohio 44106, and ^¶¶ Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44106

Diabetic retinopathy, a retinal vascular disease, is inhibited in animals treated with aminoguanidine, an inhibitor of inducible nitric-oxide synthase. This treatment also reduces retinal protein nitration, which is greater in diabetic rat retina than nondiabetic retina. As an approach to understanding the molecular mechanisms of diabetic retinopathy, we sought the identity of nitrotyrosine-containing proteins in retina from streptozotocin-induced diabetic rats and in a rat retinal Müller cell line grown in high glucose (25 mM). Anti-nitrotyrosine immunoprecipitation products from rat retina and Müller cells were analyzed by LC-MS/MS. Ten nitrated proteins in diabetic rat retina and three nitrated proteins in Müller cells grown in high glucose were identified; three additional nitrotyrosine-containing proteins were tentatively identified from diabetic retina. The identified nitrotyrosine-containing proteins participate in a variety of processes including glucose metabolism, signal transduction, and transcription/translation. Among the nitrated proteins were insulin-responsive glucose transporter type 4 (GLUT-4), which has been implicated previously in the pathogenesis of diabetes mellitus; exocyst complex component Exo70, which functions in insulin-stimulated glucose uptake of GLUT-4-containing vesicles; and fibroblast growth factor receptor 2, which influences retinal vascularization via fibroblast growth factor signaling. Nitration of tyrosine phosphorylation sites were identified in five proteins, including GLUT-4, exocyst complex component Exo70, protein-tyrosine phosphatase , sensory neuron synuclein, and inositol trisphosphate receptor 3. Quantitation of nitration and phosphorylation at common tyrosine modification sites in GLUT-4 and protein-tyrosine phosphatase from diabetic and nondiabetic animals suggests that nitration reduced tyrosine phosphorylation 2x in these proteins from diabetic retina. The present results provide new insights regarding tyrosine nitration and its potential role in the molecular mechanisms of diabetic retinopathy.