Alpha Crystallin Derived Peptide Chaperone Protects Human RPE Cells From Oxidative Injury

Abstract

Purpose: : Our previous work established that α-crystallins are anti-apoptotic. Furthermore, 19-20 amino acid sequences were identified from both αA and αB crystallins that exhibited similar chaperone function as that of full-length proteins (Sharma et al JBC 275, 3767-3771; Biochemistry 45, 3069-3076). The purpose of the present study is to examine whether these crystallin-derived peptides have anti-apoptotic functions in RPE cells.

Methods: : Protective role of α-crystallins was studied in stable ARPE-19 cell lines overexpressing full length α-crystallins. Cells were subjected to oxidative stress and cell protection was assessed by TUNEL analysis and activation of caspase 3. 19-20mer peptides of αA and αB crystallin having known chaperone function were synthesized (Neo-Peptide, MA). Early passage human fetal RPE cells were co-treated with varying doses (50-75µg/ml) of minipeptides of either αA or αB crystallin or scrambled peptides and tBH or H2O2 to induce oxidative stress. Cell death and caspase activation were determined. The uptake of mini-peptide was studied using fluorescence labeled αB crystallin mini-peptide under conditions of oxidative stress. Retinal uptake of the αB crystallin derived peptide in vivo was studied in a murine model of laser induced choroidal neovascularization (CNV).

Results: : Overexpression of αA and αB crystallins significantly increased protection to RPE cells from oxidative stress-induced cell death. RPE cells challenged with either H2O2 or tBH in the presence of either αA or αB crystallin peptides, remained viable and caspase-3 activation was severely inhibited. The uptake of labeled αB crystallin peptide increased significantly in the presence of oxidative stress vs untreated control RPE cells and most of the peptide was translocated to the nucleus. In mice, intravenous injection of labeled αB crystallin-derived peptide showed prominent localization within CNV lesions.

Conclusions: : As with full-length α-crystallins, crystallin-derived peptides offer protection from oxidative stress-induced injury. Stress causes significant translocation of crystallin peptides to the nucleus which could activate transcription factors involved in anti-apoptotic pathways.

Keywords: crystallins • protective mechanisms • oxidation/oxidative or free radical damage