Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.
OBJECTIVE
The R120G mutation of alphaB-crystallin is known to cause desmin-related myopathy, but the mechanisms underlying the formation of cataract are not clearly established. We hypothesize that alteration of protein-protein interaction between R120G alphaB-crystallin and lens intermediate filament proteins is one of the mechanisms of congenital cataract.


METHODS
Protein-protein interactions were determined by confocal fluorescence resonance energy transfer (FRET) microscopy using green fluorescence protein (GFP) as the donor and red fluorescence protein (RFP) as the acceptor. The lens vimentin gene was fused into a GFP vector and the alphaB-crystallin (WT or R120G mutant) gene was fused into the RFP vector. The donor-acceptor plasmid pairs of intermediate filament (IF)-GFP and alphaB-RFP were co-transfected into HeLa cells. After incubation, confocal fluorescence images of the transfected cells were taken. FRET was estimated by the acceptor photobleaching method. Protein-protein interaction was evaluated by FRET efficiency.


RESULTS
The confocal fluorescence images showed that the cells expressing vimentin and R120G alphaB-crystallin contained large amounts of protein aggregates while few vimentin fibers were observed. FRET efficiency analyses indicated that vimentin had a significantly greater protein-protein interaction with R120G alphaB-crystallin than with WT alphaB-crystallin.


CONCLUSIONS
Our results show that the R120G alphaB-crystallin mutant promoted vimentin aggregation through increased protein-protein interaction. This process may contribute to the formation of congenital cataract.
Song
Shuhua
S
Center for Ophthalmic Research/Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.
Hanson
Mark J
MJ
Liu
Bing-Fen
BF
Chylack
Leo T
LT
Liang
Jack J-N
JJ
D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D050505:Mutant Proteins / Q000378:metabolism; D038761:Photobleaching; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014746:Vimentin / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D038203:alpha-Crystallin B Chain; D000097573:Red Fluorescent Protein