Elastin-like polypeptide (ELP) fused to an αB crystallin mini-chaperone protects RPE cells from oxidant injury

Abstract

Purpose: In addition to being a chaperon, αB crystallin has multiple functions including inhibition of apoptosis. The purpose of this study was to investigate the protective effect of protein polymer nanoparticles decorated with αB crystallin minipeptide (residues 73-92) that protects against oxidative injury to RPE. Derived from human tropoelastin, these elastin-like polypeptide (ELP) polymers assemble nanostructures, which we hypothesized can modulate the RPE uptake, retention, and efficacy of the mini-peptide.

Methods: ELP-αB crystallin mini-chaperone (ELP- αB crystallin) fusion protein was expressed and purified. Chaperone activity of the purified ELP-αB crystallin was measured by the alcohol dehydrogenase assay. To study the protective effect of ELP-αB crystallin, primary human RPE cells were co-incubated with varying doses of ELP - αB crystallin or ELP alone and 200 µM H2O2 for 24 h. RPE cell death and caspase-3 activation induced by oxidative stress were determined by TUNEL assay and Western blot analysis. Cellular uptake was determined in ELP particles after conjugation with rhodamine and incubation of cells with 200 µM H2O2 and 5 µM labeled ELP for 24 h and analysis of confocal microscopic images. Subcellular localization of uptake was studied by transmission electron microscopy (TEM).

Results: ELP-αB crystallin mini-chaperone (ELP- αB crystallin) fusion protein was expressed and purified. Chaperone activity of the purified ELP-αB crystallin was measured by the alcohol dehydrogenase assay. To study the protective effect of ELP-αB crystallin, primary human RPE cells were co-incubated with varying doses of ELP - αB crystallin or ELP alone and 200 µM H2O2 for 24 h. RPE cell death and caspase-3 activation induced by oxidative stress were determined by TUNEL assay and Western blot analysis. Cellular uptake was determined in ELP particles after conjugation with rhodamine and incubation of cells with 200 µM H2O2 and 5 µM labeled ELP for 24 h and analysis of confocal microscopic images. Subcellular localization of uptake was studied by transmission electron microscopy (TEM).

Conclusions: ELP-αB crystallin fusion proteins are taken up efficiently by RPE cells and offer cellular protection through their intracellular actions by inhibiting caspase-3 activation.

Keywords: 488 crystallins • 426 apoptosis/cell death • 450 chaperones