Abstract
Purpose:
It has been shown that mini-chaperone, a peptide representing the chaperone binding site in αA-crystallin prevents destabilized protein aggregation. It was also shown that mini-chaperone forms amyloid fibrils. The present study was undertaken to improve mini-chaperone stability while preserving its anti-aggregation activity by fusing the flexible and solvent exposed C-terminal 163-173 region of αA-crystallin to the mini-chaperone, DFVIFLDVKHFSPEDLTVK to get a chimeric peptide (CP1) chaperone and to characterize the same
Methods:
Circular dichroism, TEM, Thioflavin T binding assays were used to characterize the peptide chaperone. The chaperone activity of the CP1 was measured with ADH, CS and αLA as client proteins. ARPE-19 cells were used to determine the anti-oxidation/anti-apoptotic activity.
Results:
Circular dichroism studies showed that unlike the beta sheet structure shown by mini-chaperone the CP-1 peptide exhibited random structure. Examination of CP-1 peptide incubated in a shaker at 37o C for 72 hrs under TEM did not show amyloid fibrils whereas theα A-mini-chaperone showed distinct fibrils. Consistent with TEM observation, Thioflavin T binding assays showed increased dye binding with mini-chaperone incubated at 37oC and subjected to shaking but not with CP1 peptide incubated under similar conditions. The chaperone activity of the CP1 peptide was comparable to that of mini-chaperone when denaturing ADH, CS and αLA were used as client proteins. Transduction of both peptide chaperones to ARPE cells showed no cytotoxic effects. The anti-oxidation assay involving H2O2 treatment of ARPE cells revealed that both original mini-chaperone and CP1 peptide have comparable cytoprotective activity against hydrogen peroxide induced oxidative damage.
Conclusions:
The present study shows that addition of C-terminal sequence 163-173 of αA-crystallin to αA-mini-chaperone influences the conformation of αA-mini-chaperone without affecting its chaperone function or cytoprotection activity.
Keywords: 450 chaperones •
488 crystallins •
634 oxidation/oxidative or free radical damage