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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-VISA (01))
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Araj, Houmam H
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University of Washington
Anatomy/Cell Biology
Schools of Medicine
United States
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Clark, John I (2016) Functional sequences in human alphaB crystallin. Biochim Biophys Acta 1860:240-5
Anderson, David M G; Floyd, Kyle A; Barnes, Stephen et al. (2015) A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging. Anal Bioanal Chem 407:2311-20
Clark, John I (2013) Self-assembly of protein aggregates in ageing disorders: the lens and cataract model. Philos Trans R Soc Lond B Biol Sci 368:20120104
Gokhin, David S; Nowak, Roberta B; Kim, Nancy E et al. (2012) Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens. PLoS One 7:e48734
Clark, Tyler J W; Houck, Scott A; Clark, John I (2012) Hemoglobin interactions with ?B crystallin: a direct test of sensitivity to protein instability. PLoS One 7:e40486
Greiling, Teri M S; Clark, John I (2012) New insights into the mechanism of lens development using zebra fish. Int Rev Cell Mol Biol 296:1-61
Qu, Bo; Landsbury, Andrew; Schönthaler, Helia Berrit et al. (2012) Evolution of the vertebrate beaded filament protein, Bfsp2; comparing the in vitro assembly properties of a ""tailed"" zebrafish Bfsp2 to its ""tailless"" human orthologue. Exp Eye Res 94:192-202
Houck, Scott A; Landsbury, Andrew; Clark, John I et al. (2011) Multiple sites in ?B-crystallin modulate its interactions with desmin filaments assembled in vitro. PLoS One 6:e25859
Houck, Scott A; Clark, John I (2010) Dynamic subunit exchange and the regulation of microtubule assembly by the stress response protein human alphaB crystallin. PLoS One 5:e11795
Greiling, Teri M S; Aose, Masamoto; Clark, John I (2010) Cell fate and differentiation of the developing ocular lens. Invest Ophthalmol Vis Sci 51:1540-6

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