April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Phosphorylation of α-crystallins: regulation of neuroprotective properties and chaperone function
Author Affiliations & Notes
  • Jennifer Dulle
    University of Michigan, Ann Arbor, MI
  • Sean Grafton
    University of Michigan, Ann Arbor, MI
  • Patrice E Fort
    University of Michigan, Ann Arbor, MI
  • Footnotes
    Commercial Relationships Jennifer Dulle, None; Sean Grafton, None; Patrice Fort, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 1044. doi:
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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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 499 diabetic retinopathy • 488 crystallins • 615 neuroprotection  
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