Research will continue on the long term goal of elucidating the structural and mechanistic bases of ?-crystallin chaperone activity, defining its contribution to the maintenance of lens transparency and understanding its role in the development of cataract. Cataract is the leading cause of blindness world-wide and its treatment remains a significant burden to the US health care system. Aging degrades lens optical properties through modifications of lens proteins which compromise their stability, promote their aggregation and disturb the protein order required for refractivity and transparency. The dominant model in the field hypothesizes a central role for the lens small heat-shock protein ?-crystallin in delaying the onset of aggregation through recognition and binding of destabilized lens proteins. Despite 20 years of progress in identification of age-related modifications and an expansive effort to define ?-crystallin chaperone mechanism, there remains a divide between in vitro models of lens protein interactions and congenital and age-related cataract phenotypes. The proposed research plan will attempt, for the first time, to challenge mechanistic in vitro hypotheses and models of lens protein stability and ?-crystallin chaperone function in the intact lens of zebrafish (Danio rerio). An outstanding team of collaborators has been recruited to enable the successful completion of this multidisciplinary plan.
Aim 1 will continue our productive studies of ?-crystallin structure and mechanism. We will test if a structural model of ?-crystallin activation, developed in the previous funding period, explains the evolutionary variation in chaperone properties and the loss of binding regulation in mutants of ?-crystallin associated with human autosomal dominant hereditary cataracts. We will use spectroscopy to investigate the structural and energetic aspects of the chaperone interaction between ?- and ?-crystallin.
Aim 2 proposes to generate transgenic zebrafish models to determine how ?-crystallin chaperone affinity is shaped by the crowded environment of the lens fiber cells and whether cataract phenotypes can be rescued by engineered ?-crystallin with designed functional properties.
By bridging the divide between crystallin stability and interactions and lens transparency, the stage will be set for developing strategies to delay or inhibit cataract formation. Furthermore, the development of zebrafish models that recapitulate aspects of human cataract will ultimately provide a platform to identify cataract-treating compounds.
|Mishra, Sanjay; Chandler, Shane A; Williams, Dewight et al. (2018) Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity. Structure 26:1116-1126.e4|
|Mishra, Sanjay; Wu, Shu-Yu; Fuller, Alexandra W et al. (2018) Loss of ?B-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart. J Biol Chem 293:740-753|
|Wu, Shu-Yu; Zou, Ping; Fuller, Alexandra W et al. (2016) Expression of Cataract-linked ?-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability. J Biol Chem 291:25387-25397|
|Koteiche, Hanane A; Claxton, Derek P; Mishra, Sanjay et al. (2015) Species-Specific Structural and Functional Divergence of ?-Crystallins: Zebrafish ?Ba- and Rodent ?A(ins)-Crystallin Encode Activated Chaperones. Biochemistry 54:5949-58|
|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|
|Zou, Ping; Wu, Shu-Yu; Koteiche, Hanane A et al. (2015) A conserved role of ?A-crystallin in the development of the zebrafish embryonic lens. Exp Eye Res 138:104-13|
|Shi, Jian; Koteiche, Hanane A; McDonald, Ezelle T et al. (2013) Cryoelectron microscopy analysis of small heat shock protein 16.5 (Hsp16.5) complexes with T4 lysozyme reveals the structural basis of multimode binding. J Biol Chem 288:4819-30|
|Yirdaw, Robel B; McHaourab, Hassane S (2012) Direct observation of T4 lysozyme hinge-bending motion by fluorescence correlation spectroscopy. Biophys J 103:1525-36|
|Mishra, Sanjay; Stein, Richard A; McHaourab, Hassane S (2012) Cataract-linked ýýD-crystallin mutants have weak affinity to lens chaperones ýý-crystallins. FEBS Lett 586:330-6|
|McHaourab, Hassane S; Lin, Yi-Lun; Spiller, Benjamin W (2012) Crystal structure of an activated variant of small heat shock protein Hsp16.5. Biochemistry 51:5105-12|
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