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.
Showing the most recent 10 out of 28 publications
