In order to understand how a pathogenic change in a gene causes disease, it is necessary to recognize a protein structure-function and a role in protein networks. We are looking for a general approach in which computational methods are used to probe the severity of protein structural perturbations caused by pathogenic mutation and to provide a basis for associating these changes with disease phenotype. The implications of such an approach to the kingdom of proteins affected by inherited and degenerative eye disease could create a universal scale to compare effects of mutations at the atomic level, thus, guiding better diagnosis of inherited disease. We successfully applied this approach in molecular modeling of retinoschisin with functional analysis of 27 mutations from 61 X-linked retinoschisis (XLRS) patients and demonstrated that mutational change in RS1 protein structure affects the phenotype of XLRS measured from electroretinogram (manuscript is ready for submission). In 10 pathogenic mutations causing choroideremia missense L550P, truncation and deletion mutants were predicted using computational modeling to be associated with a partial or total loss of the Rab excort protein 1 (REP-1). The presumptive loss of protein was confirmed by Western Blot analysis of protein from mononuclear cells and fibroblasts from CHM patients. Recently, I have applied molecular modeling to explain a functional role of three mutations in the paried box transcription factor gene, PAX2, which may cause papillorenal syndrome. In collaboration with Dr. Brian Brooks I performed molecular modeling of the DNA-PAX2 complex to show a structural effect of these missense mutations. The manuscript describing this work and in which I am a co-author, has received a good review in the PLOS Molecular Genetics and is expected to be accepted. In the experimental part of our work, we have used recombinant protein expression and purification to generate wild type and modified beta-crystallin proteins. Protein folding/misfolding, protein aggregation, subunits exchange, stability and formation of oligomeric homo- and hetero-associates in protein-protein interactions were studied. These characterizations, lead to a better understanding of the different elements of protein structure defining native interactions such as terminal extensions, globular domains and their structural parts. Recently, we demonstrated using site directed mutagenesis, protein expression/purification, limited proteolysis and molecular modeling, that the amino terminal of betaB1 extension contains structural features which position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear critical for mediating protein interactions with betaA3-crystallin. This work is done in collaboration with Dr. Paul Wingfield (NIAMS) and his laboratory. These results were presented at ARVO meeting 2009 and the manuscript is accepted for publication in Biochemistry. In collaboration with Dr. Sinha laboratory, I used molecular dynamic simulations to show that the 10-residue insertion in betaA3/A1-crystallin creates a loop at the C-terminus that sterically affects protein-protein interactions. This was confirmed by my collaborators who showed aggregation of the mutant betaA3/A1 in the lens. In addition, this study showed for the first time that betaA3/A1 is expressed in the astrocytes, has a novel function in the remodeling of the retina and is also expressed in retinal pigment epithelium cells where it shows age-related macular degeneration (AMD)-like pathology in the aging Nuc1 spontaneous mutant rats.
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