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M. Tanito, H. Haniu, H. Matsumoto, R.E. Anderson; Proteomics Analysis of 4–Hydroxynonenal–Modified Proteins in the Stages Preceding the Light–Induced Retinal Degeneration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2886.
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© ARVO (1962-2015); The Authors (2016-present)
4–Hydroxynonenal (4–HNE) is a reactive aldehyde species derived in vivo from the non–enzymatic oxidation of n–6 polyunsaturated fatty acids. We have reported that intense light exposure increases 4–HNE–protein modification in the retina in the early stages that precede photoreceptor cell apoptosis. In this work, we have used a proteomic approach to identify 4–HNE–modified retinal proteins, in order to understand the molecular mechanism underlying the retinal degeneration.
Albino rats were exposed to 5 k lux white fluorescent light for 3 h and retinas were removed 24 h later and pooled (4 rats). The levels and localization of 4–HNE–modified proteins in retinas were assessed by Western dot blots and by immunohistochemistry, respectively. 4–HNE–modified proteins were identified by 2–dimensional gel electrophoresis (2–DE) followed by Western blot, and subjected to peptide mass fingerprinting.
By dot blot analysis, total intensity of 4–HNE–modified proteins was increased 1.5–fold following exposure. By 2 independent sets of 2–DE/Western blots, more than 600 proteins and 87 4–HNE–modified protein spots were detected. After computer–assisted densitometry, 23 spots that were consistently increased by more than 1.5–fold were taken for further analysis. By mass spectrometry and peptide mass fingerprinting, 5 chaperone–, 4 energy metabolism–, 4 cell signal/kinase/channel–, 2 RNA synthesis–, and 2 structural–related proteins and others were identified. The modification appears to be specific to a particular set of proteins rather than random events on abundant proteins. By immunohistochemistry, 5 identified proteins (heat shock protein 70, glutamine synthetase 1, transducin ß1, heterogenous nuclear ribonucleoprotein A2/B1, and actin ß) were co–localized with the signals of 4–HNE–modified proteins in light–exposed retinas.
Intense light exposure increases 4–HNE–protein modifications in specific retinal proteins. These modifications may be involved in the initiation of light–induced retinal degeneration.
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