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Jennifer Dulle, Sean Grafton, Patrice E Fort; Phosphorylation of α-crystallins: regulation of neuroprotective properties and chaperone function. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1044.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that the small heat shock chaperones αA- and αB-crystallin are up-regulated in the retinas of mouse models of Type 1 diabetes. Despite up-regulation of α-crystallins, diabetic mice exhibit increased retinal cell death compared to non-diabetic mice suggesting reduced α-crystallin function. In correlation, our previous data have shown that diabetes results in decreased solubility of α-crystallins and a loss of interaction with the proapoptotic protein Bax. Additionally, post-translational modifications (PTMs) of α-crystallins are known to regulate both their oligomerization and function, thus we hypothesized that diabetes may reduce α-crystallin function by affecting the pattern of PTMs. In this investigation, we aim to understand how phosphorylation affects the function and neuroprotective mechanism of α-crystallins in retinal neurons.
In order to characterize the effect of PTMs on the neuroprotective function of α-crystallins, we overexpressed phosphomimetic and nonphosphorylatable mutants of the major phosphorylation sites of αB-crystallin in retinal neurons under diabetes-associated stress conditions. We characterized the effect of the individual and combined mutants on preventing cell death and caspase activation. We then utilized a combination of biochemical methods including solubility, oligomerization, and chaperone activity assays and immunoprecipitation with apoptotic factors to determine the phosphorylation-induced changes in conformation and function of αB-crystallin. We also analyzed the subcellular localization of the mutants by immunofluorescence and cellular fractionation.
We have found that the phosphomimetic mutant displays different neuroprotective properties than does the wild type and nonphosphorylatable αB-crystallin. In addition, we have shown that the phosphorylation state of αB-crystallin affects the conformation and function of αB-crystallin in retinal neurons by altering αB-crystalllin oligomerization, chaperone activity toward aggregated substrates, and interaction with pro-apoptotic factors.
Our work demonstrates that PTMs can regulate the neuroprotective properties of α-crystallin and as such may play a role in the reduced function of α-crystallins in diabetes. Understanding how PTMs regulate α-crystallin function in neurons will provide insight into the mechanism of retinal cell death in diabetic retinopathy.
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