A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness

Abstract

SummaryThe autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.

Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease

“…M39R rhodopsin displayed a nearly wild-type phenotype, colocalizing with a plasma membrane marker but with some evidence of mutant rhodopsin retained within the cell (Figure S8), similar to a previous study (Davies et al 2012). G51A displayed robust wild-type-like localization on the plasma membrane, consistent with a recent report (Behnen et al 2018). This comparative range of subcellular localization matched what we had previously observed in assays of rhodopsin function for these three mutations.…”

“…As the majority of disease-linked rhodopsin mutations cause the receptor to misfold (Athanasiou et al 2018), comparing the subcellular localization of rhodopsin using mammalian cells is a common technique to study protein trafficking and ER retention, and is predictive of pathogenicity (Sung et al 1991;Behnen et al 2018). To establish phenotypes in mammalian cells to compare to, we again used the rhodopsin mutations P23H, M39R, and G51A to create a gradient of phenotypic severity.…”

Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration

“…strategies (e.g., with neuroprotective factors aimed at delaying cell death) may at the most provide a longer time window for future causal treatment (75). For some retinopathies, specific compounds for effective treatment have been identified, but this usually requires large-scale screening of substances (76,77). Here, the situation is different: the specific "therapeutic compound" would target taurine homeostasis (provided its defective TAUT-mediated transport can be bypassed) to prevent retinal degeneration and potential further organ pathologies in later life.…”

Characterizing variants of unknown significance in rhodopsin: a functional genomics approach

“…Additional assays are needed to detect and distinguish other mutation classes. For example, to identify mutations that are rescued by retinal binding (Mendes, 2008;Krebs, 2010;Mendes, 2010;Behnen, 2018), the assay could be repeated in the presence of retinal (which was too toxic for use in the current transient transfection system; not shown). For the purpose of evaluating pathogenicity, general toxicity-or stress-based assays would be ideal.…”

“…The reported biochemical classes were obtained from the literature (Mendes, 2005;Rakoczy, 2011;Athanasiou, 2018). (Additional historical categorizations have also been published (Sung, 1993;Krebs, 2010), as well as classifications that integrate pharmacologic response (Behnen, 2018 Labeled variants that were previously considered VUS (L47R, G18D, G101V, and P180T) have now been demonstrated to express at pathogenic levels. Note that the variants with high expression levels are not necessarily all benign, as the assay does not detect all classes of mutations.…”