Addition of αA-Crystallin Sequence 164–173to a Mini-Chaperone DFVIFLDVKHFSPEDLTAlters the Conformation but Not the Chaperone-like Activity

摘要

It has been shown that αA-mini-chaperone, a peptide representing the chaperone binding site in αA-crystallin, prevents destabilized protein aggregation. αA-Mini-chaperone has been shown to form amyloid fibrils. This study was undertaken to improve the stability of αA-mini-chaperone while preserving its anti-aggregation activity by fusing the flexible and solvent-exposed C-terminal 164–173 region of αA-crystallin to the mini-chaperone sequence DFVIFLDVKHFSPEDLT. The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTSAPSS (designated CP1), was characterized. Circular dichroism studies showed that unlike αA-mini-chaperone with its β-sheet structure, the CP1 peptide exhibited a random structure. Transmission electron microscopy (TEM) examination of the CP1 peptide incubated in a shaker at 37 °C for 72 h did not reveal amyloid fibrils, whereas αA-mini-chaperone showed distinct fibrils. Consistent with TEM observation, the thioflavin T binding assay showed an increased level of dye binding in the mini-chaperone incubated at 37 °C and subjected to shaking but not of the CP1peptide incubated under similar conditions. The chaperone activityof the CP1 peptide was comparable to that of αA-mini-chaperoneagainst denaturing alcohol dehydrogenase, citrate synthase, and α-lactalbumin.Transduction of both peptide chaperones to COS-7 cells showed no cytotoxiceffects. The antioxidation assay involving the H2O2 treatment of COS-7 cells revealed that αA-mini-chaperoneand the CP1 peptide have comparable cytoprotective properties againstH2O2-induced oxidative damage in COS-7 cells.This study therefore shows that the addition of C-terminal sequence164–173 of αA-crystallin to αA-mini-chaperone influencesthe conformation of αA-mini-chaperone without affecting itschaperone function or cytoprotective activity.