Interaction of alpha-crystallin with cholesterol bilayer domains in cataract formation

Project Summary: This competitive renewal grant is a request for support in further elucidating the mechanism that initiates α-crystallin (αABc) binding to the lens membrane, causing cataract formation. αABc is a major protein comprising two subunits, αA-crystallin (αAc) and αB-crystallin (αBc), in roughly a 3:1 ratio of αAc to αBc making αABc heteropolymer found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of αABc are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound αABc, accompanied by increased light scattering and cataract formation. The mechanism that initiates αABc binding to the lens membrane causing cataract formation is unknown; therefore, determining the mechanism is crucial in preventing and reversing cataract development. The broad objective of this proposal is to understand the role of cholesterol (Chol) and cholesterol bilayer domains (CBDs) in the fiber-cell plasma membranes of the eye lens in αABc membrane binding. We have published twelve papers under the Aims of the currently funded proposal, and the findings from the funded project have directed us to new, more detailed studies that we proposed in the new Aims of this competitive renewal grant. Our significant findings under the funded Aims show that 1) Lipids (phospholipids and sphingolipids) and Chol composition strongly modulate αABc binding to membranes, and 2) Chol and CBDs inhibit the binding of αABc to membranes. Earlier studies reported that deamidation of αAc and αBc, i.e., αAc-N101D, αAc-N123D, αAc-Q147E, αBc-N78D, and αBc-N146D in aging and cataract development, genetic mutation of αAc and αBc, i.e., αAc-R116C, αAc-R49C, αAc-G98R, αBc-R120G and αBc-D140N causing cataract, and the oxidation of lens lipids and Chol as a possible cause of cataract however the underlying mechanism of cataract formation is unknown. Building upon the knowledge acquired during the current grant period and earlier studies, we hypothesize that deamidation and mutation of αABc and oxidation of lipids and Chol promote αABc binding to the membrane, and Chol and CBDs inhibit such binding and protect against cataract formation and progression. We will use the innovative state-of-the-art electron paramagnetic resonance (EPR) and atomic force microscopy (AFM) methods to test our hypothesis with three specific Aims: 1) Determine the effects of deamidation of αAc and αBc in αABc membrane binding, 2) Elucidate the effects of the genetic mutations of αAc and αBc in the binding of αABc to the membrane, and 3) Determine the effects of oxidation of lipids and Chol in the binding of αAc, αBc, and αABc to the membranes. The analysis will include the donor's health history, sex, and race. The findings from this research will generate fundamental information on the molecular level that will explain the mechanism of αAc, αBc, and αABc binding to the lens membranes, leading us to understand the causes and mechanisms of congenital, early onset, and age-related cataract.