Integrated in silico and in vitro characterization of Rhodopsin mutations and molecular mechanisms activated in photoreceptor cell death

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 identified in the rhodopsin gene (RHO) as cause of the Autosomal Dominant form of the disease (ADRP). 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 and the cell death pathways activated by mutant RHO are obscure, hampering 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. Subcellular localization and effects of retinal as chaperone in the same mutants were characterized in vitro in COS-7 cells. Molecular death pathways activated by RHO mutation were studied in vivo in transgenic and knock-in mice bearing the P23H mutation. 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. 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 as well as different abilities of the mutated proteins to be affected by 9-cis retinal. We characterized ER-stress pathways and calpain pathway activation in P23H mutant retinas. We defined the different contributions of these pathways by in vivo treatments with specific drugs blocking either ER-stress or calpains. Conclusions: The in silico and in vitro 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. We also characterized cell death pathways activated by RHO mutants. This knowledge will be our starting point for an in silico screen of chaperone molecules to be tested in vivo