Defining the Role of a Novel Protein Death Domain in Human RPE Cells.

Abstract

Abstract: : Purpose: The purpose of this study is to define the role of the SH3BP4 death domain in RPE cells with respect to subcellular targeting and intracellular function. Methods: Subconfluent cultures of ARPE–19 cells were transfected with either GFP or myc–tagged SH3BP4 constructs of varying lengths. Cells were fixed 24, 48 and 72 hours after transfection; myc–tagged proteins were stained for using a myc–epitope specific antibody and nuclei were stained with Sytox–orange. Cells were examined using epifluorescence and confocal microscopy. Additionally, cell layers were dissolved in 2%SDS in protease inhibitor buffer and levels of transfected proteins were quantitated using Western analysis. Results: Full length constructs expressing SH3BP4 fusion proteins target to membranes and nuclei, the same subcellular fractions as endogenous protein. Truncations of SH3BP4 showed a unique sub–organelle distribution that differed depending on the domains being expressed. Sytox orange was used to stain for nuclear detail in transiently transfected ARPE cells and results showed that over–expression of full length SH3BP4 resulted in dramatic nuclear condensation compared to control cultures. Likewise, over–expression of truncated SH3BP4 containing the death domain also showed an increase in cells with condensed nuclei compared to non–expressing neighboring cells or controls. When transfected RPE cells were stressed with the pro–oxidant t–butyl hydroperoxide, a greater percentage of cells over–expressing SH3BP4 underwent nuclear condensation at earlier time points compared non–expressing neighboring cells or control cultures. Conclusions:The death domain region of SH3BP4 is critical for specific sub–organelle targeting of the protein. Over–expression of SH3BP4 in RPE cells results in the majority of the cells undergoing nuclear condensation within 48–72 hours after transfection. Nuclear condensation is a marker for apoptosis activity and over–expression of SH3BP4 may influence programmed cell death.

Keywords: apoptosis/cell death • retinal pigment epithelium • protein structure/function