Human alphaBeta crystallin is the archetype for small heat shock proteins, sHSP, that are involved in protein aggregation and filament assembly diseases including cataracts, neurodegeneration, cardiomyopathy and desmin related myopathy. Interactions between alphaBeta crystallin are necessary for normal filament assembly and organization of crystallins in lens cells.
In aim 1, characterization of the interactive sites for subunit assembly, for cytoskeletal proteins and for target peptides on human alphaBeta crystallin, the peptide sequences of the interactive domains on human alphaBeta crystallin will be identified using a protein multipin arrays. The affinities between the interactive domains will be quantified using surface plasmon resonance (SPR) and characterized functionally using in vitro and in vivo assays for chaperone activity. The results are expected to provide new information on the structural basis for the assembly of sHSP subunits to functional complexes and for their interaction with chaperone target proteins and with cellular filaments and cytoskeletal elements.
In aim 2, in vivo evaluation of retina - lens relationships that may influence development and maintenance of lens transparency in transgenic mice, the historical hypothesis that a lens - retina relationship is important for normal development of lens cell transparency will be studied. Electroretinograms (ERG) and digital slit lamp recordings of opacity in selected animal models will quantify transparency with retinal function during the development of the lens and during loss of transparency in models for cataract formation. Lastly, the hypothesis that lens cytoskeleton provides a scaffold for development and maintenance of transparent lens fiber structure will be investigated in aim 3, observe the cellular organization of major structural proteins in differentiating lens fibers during development of lens transparency and during loss of transparency in the selenite rat and in selected transgenic mouse models using confocal microscopy and electron microscopy (EM). The patterns and distribution of the cytoskeleton and crystallins during differentiation of transparent lens fibers will be investigated using electron microscopy and confocal immunocytochemistry.
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