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Valeria Marigo, Petra Behnen, Angelo Felline, Francesca Fanelli; Classification of Rhodopsin mutations by integrated in silico and in vitro analyses for screening of chaperon molecules to rescue misfolding. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2597.
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© ARVO (1962-2015); The Authors (2016-present)
About 140 point mutations were identified in the rhodopsin gene (RHO) as cause of Autosomal Dominant Retinitis Pigmentosa (ADRP), a genetic degenerative disease causing blindness in later life. 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Characterization of the structural and molecular features of such mutants will support the development of rational drug design.
Wild type RHO and 33 RHO mutations were analyzed in silico either in the rhodopsin form bound to retinal or in the opsin form by thermal unfolding simulations combined with the graph-based Protein Structure Network (PSN) analysis. The same mutants were expressed in vitro in COS-7 cells either in the absence or presence of 9-cis retinal. The subcellular localization was analyzed by immunofluorescence. Retention in the endoplasmic reticulum (ER) was assessed by co-localization of RHO with calnexin and calculation of the Pearson Correlation Coefficient (PCC).
In silico studies revealed that the selected ADRP RHO mutations impair hubs and links that are stable in the structure network of the wt protein. Remarkably, for both the rhodopsin and opsin forms, a computational index accounting for such structural behavior correlates linearly with the PCC measuring ER retention of RHO, i.e. the higher the loss of stable hubs and links, the higher PCC. The PSN-based indices allowed the sub-division of the mutants in four clusters that separate mutants that improve their structure when bound to retinal from those that do not, in perfect line with the in vitro results. While in retinal-responsive mutants the structural perturbations of opsin are released in the presence of retinal, retinal not-responsive mutants, even in the presence of retinal, retain a marked perturbation just in those links that characterize the retinal binding site. Such perturbation is associated with a structural deformation of the retinal itself.
Integrated cellular and atomic levels of analyses allowed a novel classification of ADRP RHO mutants. We developed a PCC index to evaluate the effect of retinal on protein folding and localization at the plasma membrane. We are now starting in silico screenings of compounds able to bind the retinal site and act as chaperones.
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