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Geeng-Fu Jang, Alecia Cutler, Lei Zhang, John Crabb, Heidi Stoehr, John Crabb, Bela Anand-Apte; Quantitative Proteomic Analysis of TIMP3 Dysfunction. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1575.
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© ARVO (1962-2015); The Authors (2016-present)
Polymorphisms in tissue inhibitor of metalloproteinase 3 (TIMP3) have been associated with Sorsby fundus dystrophy (SFD) and age-related macular degeneration. Toward a molecular understanding of TIMP3 dysfunction, we pursued quantitative proteomic analyses of the retina and choroid from TIMP-3 knockout (KO) mice and knockin (KI) mice expressing the TIMP3 S156C mutation that causes SFD.
Soluble proteins from isolated retinas and choroid-containing posterior globes from TIMP3 KO mice (n = 5 mice), KI homozygotes (n = 5), KI heterozygotes (n = 4), and wild-type mice (n = 7) were quantified by iTRAQ technology. Protein was digested with trypsin, peptides labeled with iTRAQ tags, fractionated by strong cation exchange chromatography, and peptides were analyzed by LC MS/MS. Proteins were identified using the Swiss-Protein database and quantified using code written in R. Proteins quantified with ≥ 2 unique peptides/protein in ≥ 3 mice/strain were considered significantly altered if average ratios relative to wild-type tissues were above or below the mean ≥ 1 SD and p values ≤ 0.05.
About 284 proteins were quantified per mouse strain. More proteins were altered in the choroid fraction (n = 44) than in the retina (n = 8), with the majority detected in TIMP3 KO mice. More proteins were elevated in KO mice than decreased while very few proteins were elevated in KI mice and none were common to those elevated in KO mice. All TIMP3 KO and KI mouse strains exhibited significantly reduced amounts of crystallins in both the retina and choroid fraction, including α-crystallin B, β-crystallin B2 and α-crystallin A. Homozygous KI mice also exhibited significantly reduced levels of several collagens, fibrilin-1 and TGFβ-induced ig-h3 in the choroid fraction.
Protein changes observed in both TIMP3 KO and KI mice are more likely due to TIMP3 dysfunction than collateral genetic alterations. Our results suggest lower crystallin levels are a consequence of TIMP3 dysfunction. Crystallins are inhibitors of apoptosis. Reduced crystallins may facilitate inflammatory responses and cellular degeneration in TIMP3 dysfunctional animals, including heterozygous TIMP3 KI mice which exhibit an SFD-like phenotype. Other proteins decreased in homozygous KI mice implicate dysregulation of extracellular matrix homeostasis.
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