The long-term objective of this proposal is to determine the molecular mechanisms by which modifications of the ?-crystallins (genetic or age-onset), lead to cataract formation. Our approach is to determine the processes that transform the highly soluble ?-crystallins into light-scattering elements, using a variety of biophysical an biochemical techniques. Included in it is an understanding of the structures appearing in the condensed phase, such as amorphous aggregates, fibers, gels or crystals. Such light scattering elements can be protein-only aggregates, or proteins associated with other cellular components, such as lipid membranes. Based on the current state of our knowledge, we hypothesize that: Modifications of the ?-crystallins alter molecular interactions and lead to either (a) self-association (altered homologous interactions) or (b) association with other components (e.g., other crystallins or membranes, i.e., altered heterologous interactions). Such changes result in the formation of condensed phases or light scattering elements. We will continue to investigate human ?D-crystallin (HGD), but will also examine ?C- and ?S-crystallins (HGC and HGS), which are the only other ?-crystallins expressed in the human lens. We expect these studies to provide a broader, more comprehensive knowledge of ?-crystallin interactions under a variety of conditions and define their role in the formation of light scattering elements. To achieve our long-term goals we propose the following Specific Aims: (1) determine the solubility and other thermodynamic and spectroscopic properties of wild-type HGC and selected, cataract-associated mutants of HGC and HGD, including their interactions with model membranes. (2) Examine the effect of specific post-translational modifications (PTMs) on the solubility and other thermodynamic and spectroscopic properties of HGD and HGS. The PTMs to be examined are: Deamidation (HGS), Cys-methylation (HGD and HGS), and acetylation (HGD). (3) Examine the polymerization and formation of assemblies in HGD during unfolding, mediated by specific residues identified by computational studies. The proposed experiments will identify the molecular bases of light scattering and opacity due to specific ?-crystallin modifications in the lens, and should aid in the development of therapeutic measures.
Age-onset cataract affects millions in our aging population, and at the very least, leads to a decline in the quality of life. Childhood cataract is a challengeto treat, and the growing child needs frequent surgical interventions. This proposal seeks to elucidate the mechanistic basis of lens opacity in both these types of cataract, and is a necessary first step towards delaying or preventing the opacity and restoring vision.
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