Integrated in silico and in vitro characterization of Rhodopsin mutations causing RP4

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were discovered in the rhodopsin gene (RHO). RHO belongs to the G protein Coupled Receptor superfamily of seven-transmembrane proteins. The vast majority of the rhodopsin mutations cause the Autosomal Dominant form (ADRP) of the disease. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants are obscure, which hampers rational drug design. Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. In order to define levels of expression and differences in post-translational modifications of the mutants compared to the wild type, the proteins were analyzed by Western blotting. Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. We could define a number of computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO. We also defined different abilities of the mutated proteins to be affected by 9-cis retinal. Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. This knowledge will be our starting point for the choice of a number of mutations to be used to reveal therapeutic effect of chaperone molecules.