Abstract
Purpose :
Earlier studies from our lab have shown that the molecular chaperone protein αA-crystallin demonstrates neuroprotective properties that are regulated by S/T148 phosphorylation. While this phosphorylation has been implicated in the anti-apoptotic activity of αA-crystallin, its influence on the structural and functional biochemistry of the protein remains unclear. The present study sought to characterize the effects of S/T148 phosphorylation on the biochemical properties of αA-crystallin, with emphasis on its solubility, oligomeric structure, and chaperone function.
Methods :
Differentiated R28 retinal neurons were transfected with plasmids encoding expression of either Wild-type (WT), phosphomimetic (148D), or non-phosphorylatable (148A) mutants of αA-crystallin. Protein solubility was then assessed under normal or metabolic stress conditions including serum starvation and diabetes-like conditions using the triton solubility method. Additionally, purified recombinant aA-crystallin proteins (WT, 148D, and 148A) were used to assess the impact of the phosphorylation on the oligomerization and chaperone function using Native gel electrophoresis and chaperone activity assays.
Results :
Protein solubility was measured as a ratio of insoluble αA-crystallin relative to the total content for each construct and condition. While no differences were observed under normal condition and short metabolic stress, longer stress duration revealed a dramatic loss of solubility of the non-phosphorylatable mutant (T148A) specifically. Conversely, the phosphomimetic mutant even showed an increased solubility when compared to the WT protein (p < .05). Consistent with changes in solubility, analysis of the recombinant proteins using native gel analysis revealed a shift towards smaller-sized oligomers for the 148D mutant, and towards larger-sized oligomers for the 148A mutant respectively. Chaperone function analysis of the recombinant proteins also confirmed an increased chaperone function of the phosphomimetic aA-crystallin mutant as opposed to the non-phosphorylatable mutant.
Conclusions :
The results of this study strongly support a critical role for T148 phosphorylation on the structural and functional biochemistry of αA-crystallin, shedding light on the mechanisms of regulation of the anti-apoptotic role of αA-crystallin.
This is a 2020 ARVO Annual Meeting abstract.