Abstract
Purpose: :
To investigate protein aggregation of T5P γC-crystallin and protein-protein interaction with αA- crystallin.
Methods: :
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.
Results: :
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.
Conclusions: :
Our results demonstrate that αA- and γC-crystallin can rescue T5P γC-crystallin from aggregation. The mechanism is through increased protein-protein interaction.
Keywords: crystallins • microscopy: light/fluorescence/immunohistochemistry • mutations