Our long-term objectives are to understand how specific post-translational chemical modifications and genetic mutations of the gamma-crystallin proteins lead to lens opacity. We hypothesize that not all protein modifications are deleterious - only those that lead to increased light scattering. Our strategy designed to test this hypothesis is as follows: We will model the cataractogenicity of a given modification or mutation of a gamma-crystallin in vitro, by introducing the required change into the protein and determining the effect on the formation of protein aggregates, liquid droplets containing protein-rich and protein-poor phases, and protein crystals (i.e., protein-derived condensed phases). Protein modifications that enhance the formation of such condensed phases (all of which lead to increased light scattering) will be potentially cataractogenic. We will compare the phase behavior (liquid-liquid and solid-liquid phase separation) of native and modified gamma-crystallins to evaluate if a given modification enhances or suppresses the formation of condensates. In parallel, we will also examine protein structure and conformation using biochemical and spectroscopic methods (chromatography, HPLC, gel electrophoresis, Raman spectroscopy, circular dichroism, fluorescence, and x-ray crystallography). We propose the following Specific Aims: (1) Evaluate which physiologically relevant chemical modifications of the 7 crystallins found enhance the formation of condensed phases - (1 a) Examine the effect of modifications of cysteine residues (i.e., thiolations) with (i) glutathione (ii) cysteine (iii) cysteinylglycine and (iv) gamma-glutamyl cysteine, (1b) Examine the effect of deamidation of asparagine and glutamine residues, and (1c) Examine the effect of truncations of the 7 crystallin polypeptide chain (2) Evaluate which of the genetic variants of the gamma-crystallins enhance the formation of condensed phases - (2a) Determine the mechanisms of opacification due to mutant proteins expressed from gamma-crystallin genes associated (in the literature) with human or animal model cataracts, (2b) Determine what effect the mutant proteins expressed from gamma-crystallin gene variants due to single-nucleotide polymorphisms, have an effect on opacification. This work is designed to provide plausible mechanisms for lens opacification due to gamma-crystallin modifications and mutations. Understanding these mechanisms is an essential first step towards the development of reagents to delay the onset of opacities due to (a) the commonly encountered age-onset cataract and (b) the rare nuclear opacities of genetic origin seen in children and young adults. In view of the recent evidence in the literature that suggests that age-onset cataract may have a genetic component, the studies proposed here have the potential for predicting the susceptibility of individuals towards this disease due to normal variants of the gamma-crystallins in the general populations.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY010535-09A1
Application #
6589857
Study Section
Special Emphasis Panel (ZRG1-SSS-P (06))
Program Officer
Liberman, Ellen S
Project Start
1994-06-01
Project End
2008-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
9
Fiscal Year
2003
Total Cost
$326,000
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Ramirez, Lisa; Shekhtman, Alexander; Pande, Jayanti (2018) Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin. Biochemistry 57:2775-2785
Mallik, Prabhat K; Shi, Hua; Pande, Jayanti (2017) RNA aptamers targeted for human ?A-crystallin do not bind ?B-crystallin, and spare the ?-crystallin domain. Biochem Biophys Res Commun 491:423-428
Dixit, Karuna; Pande, Ajay; Pande, Jayanti et al. (2016) Nuclear Magnetic Resonance Structure of a Major Lens Protein, Human ?C-Crystallin: Role of the Dipole Moment in Protein Solubility. Biochemistry 55:3136-49
Banerjee, Priya R; Pande, Ajay; Shekhtman, Alexander et al. (2015) Molecular mechanism of the chaperone function of mini-?-crystallin, a 19-residue peptide of human ?-crystallin. Biochemistry 54:505-15
Pande, Ajay; Mokhor, Natalya; Pande, Jayanti (2015) Deamidation of Human ?S-Crystallin Increases Attractive Protein Interactions: Implications for Cataract. Biochemistry 54:4890-9
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; 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
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
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

Showing the most recent 10 out of 28 publications