Abstract
Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. α-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with α-crystallin may help to delay disease onset. The purpose of this study was to determine if αB-crystallin subunits containing a cell penetration peptide (gC-tagged αB-crystallin) facilitate the uptake of wild type αA-crystallin (WT-αA) in lens and retina. Recombinant human αB-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-αB). Recombinant gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37°C for 4 hours to allow for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ cultures. Similarly, crystallin mixtures were injected into the vitreous of rat eyes. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity assays were performed on α-crystallins ratios showing optimal uptake using chemically-induced or heat induced substrate aggregation assays. As determined by flow cytometry, a ratio of 1:5 for gC-αB to WT-αA was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-αB to WT-αA (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-αB to WT-αA (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of α-crystallin were found in lens and retina following intravitreal injection of homo- and hetero-oligomers in rats.
Highlights
ΑA and αB-Crystallin, the major proteins found in the lens are members of the small heat shock protein family [1,2,3]
Flow cytometric analysis of ARPE-19 cells treated with hetero-oligomers with and without a cell penetration peptides (CPP) tag indicated a substantial uptake at the 5:1 ratio
We identified a new CPP that when tagged to αB greatly enhanced the uptake of the protein in human lens epithelial cell (HLE-B3) without negatively affecting its cytoprotective activity, the ability to form high molecular weight complexes, or chaperone-like doi:10.1371/journal.pone.0137659.g005
Summary
ΑA and αB-Crystallin, the major proteins found in the lens are members of the small heat shock protein (sHSP) family [1,2,3]. A protein aggregation disease of the lens, is hypothesized to occur when levels of soluble α-crystallins are depleted [7]. Previous studies have identified small molecules that bind aggregating proteins and reduce the amount of insoluble aggregates; while others have characterized peptides from sHSP that prevent protein aggregation [9,10,11,12,13]. While these approaches have resulted in varying success, we have hypothesized that replenishment of full-length α-crystallin in the lens will delay the onset of cataract by preventing protein aggregation. We and others have previously shown these proteins prevent stress induced apoptosis [14,15,16,17,18]
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