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. Proposed here are studies on modified ?-crystallins known to be associated with either genetic or age-onset cataract.
In Aim 1 interactions between modified ?-crystallins alone will be examined (self-association), and in Aim 2 interactions of modified ?-crystallins with ?-crystallin will be examined. Most studies to date have been of the first type and have led to a better understanding of how modified ?-crystallins self-associate and form "condensed phases" which are responsible for light scattering and opacity. The second type of interaction has recently emerged as being equally important in maintaining lens transparency. Based on current knowledge, we propose the following hypothesis: Modifications of the ?-crystallins alter the protein-protein interactions and lead to either (a) self- association of mutant proteins to yield a variety of condensed phases or (b) a change in the net attractive interactions between mutant ?-crystallins and ?-crystallin. Experiments in Aim 1 will compare the thermodynamic phase diagrams, the low and high-resolution structures (using u.v.-visible, circular dichroism, fluorescence, FTIR, Raman and NMR), and stabilities of the modified forms of human ?D-, ?C-, and ?S- crystallins, with those of the wild-type protein. Phase diagrams provide the boundary conditions for the formation of condensed phases which lead to light scattering and opacity. Therefore, these studies and light scattering measurements (static and dynamic) are an integral part of the proposed studies.
In Aim 2, the interactions of those modified forms of ?-crystallins which do not self-associate will be examined with native ?-crystallin, also using similar techniques. Thus, the proposed experiments will identify molecular mechanisms that lead to light scattering and opacity due to ?-crystallin modifications in the lens, and should provide a comprehensive understanding of both forms of cataract disease - genetic, as well as age-onset cataract.
Cataracts are the leading cause of blindness worldwide and constitute a major expense to the health care system. This proposal investigates how genetic mutations and protein- modifications due to aging, lead to cataract. The results should help devise effective preventive or therapeutic measures.
|Bharat, Somireddy Venkata; Shekhtman, Alexander; Pande, Jayanti (2014) The cataract-associated V41M mutant of human ?S-crystallin shows specific structural changes that directly enhance local surface hydrophobicity. Biochem Biophys Res Commun 443:110-4|
|Banerjee, Priya R; Pande, Ajay; Patrosz, Julita et al. (2011) Cataract-associated mutant E107A of human gammaD-crystallin shows increased attraction to alpha-crystallin and enhanced light scattering. Proc Natl Acad Sci U S A 108:574-9|
|Banerjee, Priya R; Puttamadappa, Shadakshara S; Pande, Ajay et al. (2011) Increased hydrophobicity and decreased backbone flexibility explain the lower solubility of a cataract-linked mutant of ?D-crystallin. J Mol Biol 412:647-59|
|Ghosh, Kalyan S; Pande, Ajay; Pande, Jayanti (2011) Binding of ?-crystallin substrate prevents the binding of copper and zinc ions to the molecular chaperone ?-crystallin. Biochemistry 50:3279-81|
|Danysh, Brian P; Patel, Tapan P; Czymmek, Kirk J et al. (2010) Characterizing molecular diffusion in the lens capsule. Matrix Biol 29:228-36|
|Pande, Ajay; Ghosh, Kalyan S; Banerjee, Priya R et al. (2010) Increase in surface hydrophobicity of the cataract-associated P23T mutant of human gammaD-crystallin is responsible for its dramatically lower, retrograde solubility. Biochemistry 49:6122-9|
|Pande, Ajay; Gillot, Darnelle; Pande, Jayanti (2009) The cataract-associated R14C mutant of human gamma D-crystallin shows a variety of intermolecular disulfide cross-links: a Raman spectroscopic study. Biochemistry 48:4937-45|
|Pande, Ajay; Zhang, Jianchao; Banerjee, Priya R et al. (2009) NMR study of the cataract-linked P23T mutant of human gammaD-crystallin shows minor changes in hydrophobic patches that reflect its retrograde solubility. Biochem Biophys Res Commun 382:196-9|
|Wu, Yalin; Fishkin, Nathan E; Pande, Ajay et al. (2009) Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease. J Biol Chem 284:20155-66|
|McManus, Jennifer J; Lomakin, Aleksey; Ogun, Olutayo et al. (2007) Altered phase diagram due to a single point mutation in human gammaD-crystallin. Proc Natl Acad Sci U S A 104:16856-61|
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