Abstract
Purpose :
Deletion of 54-61 residues in αB-crystallin (αBΔ54-61) results in a ten-fold activation of the chaperone-like function of the protein. The present study was undertaken to explore the reasons behind the dramatic increase in the chaperone-like activity of αBΔ54-61.
Methods :
The critical structural features viz hydrophobicity, stability, and molecular size, that govern the chaperone-like function of the WT and mutant crystallins were compared. Chemical cross-linking of the WT and mutant protein oligomers with isotope-labeled (D0 and D4) BS2G crosslinker followed by mass spec analysis was carried out to find out the differences in the oligomeric organization of the subunits. The interaction of the aggregating substrates with crystallins was analyzed using a multi-angle light scattering detector. Cell integrity assay was performed to assess the cytoprotective ability of the crystallins in sodium selenite-challenged adult retinal pigment epithelial (ARPE-19) cells.
Results :
Compared to WT, the deletion mutant exhibits increased surface hydrophobicity, reduced stability and increased susceptibility to digestion by trypsin suggesting of loosely packed oligomers. The WT protein had a more compact structure and showed resistance to increase in size upon binding to a substrate protein. The deletion mutant demonstrated increased binding affinity towards unfolding substrates with a continued increase in complex size with time. Cross-linking study showed an altered subunit interactions in the mutant oligomer compared to WT protein. Cytotoxicity studies showed that the mutant protein has higher anti-apoptotic activity than the wild-type in oxidatively stressed cells.
Conclusions :
Our study shows that increased hydrophobicity, altered subunit assembly, and enhanced affinity towards the substrate proteins might have contributed towards the ten-fold activation of chaperone-like function of the αBΔ54-61 protein.
This is a 2020 ARVO Annual Meeting abstract.