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 (serum starvation) 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, and normalized to WT. While no differences were observed under normal and short metabolic stress conditions, longer stress duration revealed differences in solubility, with 148A becoming dramatically more insoluble than 148D (p<0.01, n=6), and 148D becoming slightly (though not significantly) more soluble than WT αA-crystallin. Consistent with changes in solubility, analysis of the recombinant proteins using native gel analysis revealed a shift towards smaller-size oligomers for the 148D mutant, and towards larger-size oligomers for the 148A mutant respectively. Chaperone function analysis of the recombinant proteins also confirmed a significantly increased chaperone activity of the phosphomimetic mutant compared to the WT and non-phosphorylatable mutant (IC50=4.4ug (148D) vs 8ug (WT) and 7.1ug (148A).
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 aA-crystallin.
This is a 2021 ARVO Annual Meeting abstract.