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J. J. Liang, B.-F. Liu, M. Hanson, S. Song; Confocal Fluorescence Microscopy Studies of Human Lens T5P C-Crystallin Mutant. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4088.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate protein aggregation of T5P γC-crystallin and protein-protein interaction with αA- crystallin.
Crystallin genes (T5PCRYGC, CRYGC, CRYAA) were fused to either green fluorescence protein (GFP) or red fluorescence protein (RFP) vector. Donor-acceptor plasmid pairs, such as T5PγC-GFP and αA-RFP, were cotransfected into HeLa cells. Confocal fluorescence images of the cultured cells were taken. Protein-protein interaction was evaluated by fluorescence resonance energy transfer (FRET) between the donor and acceptor.
The confocal fluorescence images show that T5P γC-crystallin expression contains a large amount of protein aggregates but co-expression with either γC- or αA-crystallin reduces the aggregation considerably. FRET determination indicates that T5PγC-T5PγC shows less protein-protein interaction than either T5PγC-γC or T5PγC-αA.
Our results demonstrate that αA- and γC-crystallin can rescue T5P γC-crystallin from aggregation. The mechanism is through increased protein-protein interaction.
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