Protein–protein interactions involving congenital cataract T5P γC-crystallin mutant: A confocal fluorescence microscopy study

Abstract

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects proteinprotein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP-RFP (donor-acceptor) plasmid pairs was cotransfected into HeLa cells: γC-γC, γC-γCT5P, γCT5P-γCT5P, αA-γC, and αAγCT5P. After culture, confocal fluorescence cell images were taken. Protein-protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P γC-crystallin contain many protein aggregates, but cells co-expressing with either γC-or αA-crystallin reduce the aggregation considerably. FRET determination indicates that γCT5P-γCT5P shows less protein-protein interaction than either γC-γC or γC-γCT5P. Cotransfection with αA-crystallin (αA-γC orαA-T5PγC) increases nFRET compared with γC-γC or γC-T5PγC. Our results demonstrate that T5PγC-crystallin shows more protein aggregates and less protein-protein interaction than WTγC-crystallin. Chaperone αA-crystallin can rescue T5P γC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein-protein interactions and increased aggregation from an insufficient amount of α-crystallin for protection.

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens

“…Among the three missense mutations (p.Thr5Pro, p.Arg48His, and p.Arg168Trp) in γC-crystallin, the effect of p.Thr5Pro on γC-crystallin has been well characterized in vitro by Liang's and several other groups. They showed that the mutation significantly impaired the secondary and tertiary structures, decreased the solubility and thermal stability of γC-crystallin [Fu and Liang, 2002] and affected its interaction with α-crystallin thereby [Fu and Liang, 2003;Liu et al, 2008]. Unlike the significant decrease in the regular secondary structure content by the p.Thr5Pro mutation [Fu and Liang, 2002], both structural modeling and spectroscopic analysis indicate that the p.Gly129Cys mutation did not affect the native secondary structure of γC-crystallin.…”

“…It is worth noting that α-crystallin in the lens acts as a molecular chaperone to protect misfolded proteins against aggregation [Horwitz, 1992]. Although α-crystallin could reduce the extent of aggregation, previous studies also show that the cataract-linked γ-crystallin mutants may affect α-crystallin function by aberrant protein-protein interactions [Banerjee et al, 2011;Liu et al, 2008]. It will be interesting to further investigate whether the p.Gly129Cys mutant also impairs or overloads α-crystallin function.…”

“…Among them, the molecular mechanism caused by p.Thr5Pro has been thoroughly studied at the protein level in the past 10 years Liang, 2002, 2003;Liu et al, 2008;Talla et al, 2006], and the deleterious effect of the five-base insertion is investigated using the transgenic mouse model in a recent work . In this study, we identified a novel p.Gly129Cys mutation of γC-crystallin in a Chinese threegeneration pedigree with autosomal dominant congenital cataract.…”

Cell Penetration Peptides for Enhanced Entry of αB-Crystallin into Lens Cells

“…Several reported cell lines expressing aggregation prone protein mutants and animal models with aggregation prone rhodopsin or c-crystallin mutants could be used to characterize a-crystallin as a therapeutic agent. [38][39][40] The fact that we have made a gC-aB that can easily be delivered to cells will allow for the characterization of a-crystallin in this setting. These models will also allow for characterization of any effect that the CPP has on in vivo use of a-crystallin.…”

Protein misfolding and aggregation in cataract disease and prospects for prevention

“…The Thr5Pro substitution in human γC-crystallin affects stability and crystallin-crystallin interactions [67–69]. Human γS-crystallin mutant Gly18Val lowers the transition midpoint of unfolding in GuHCl and reduces stability under conditions of heat denaturation [70].…”