Metabolomics Effects of Folding Correction in Retinitis Pigmentosa Rhodopsin Mutant P23A.

Retinitis pigmentosa (RP) is a hereditary retinal degeneration disorder often caused by mutations in the rhodopsin gene, leading to photoreceptor death and vision loss. While structural misfolding of rhodopsin is a known contributor to disease pathology, the mechanisms of its cellular and in particular metabolic consequences are poorly understood. To study the direct effects of rhodopsin misfolding and structural rescue on cellular metabolism, we used the P23A mutant and its N2C/D282C stabilized counterpart as a structural tool to assess how differences in folding stability relate to measurable changes at the metabolite level. The engineered cysteine pair allows the formation of a disulfide bond restoring structural integrity and reinforcing the stable seven-transmembrane bundle. We used untargeted Gas Chromatography-Mass Spectrometry (GC-MS) metabolomics analysis conducted in inducible rhodopsin-expressing cell lines, providing a broad and general profiling of metabolic pathway alterations in response to the expression of RP mutants and their structurally rescued counterparts. Principal component analysis, hierarchical clustering, and K-means clustering revealed distinct metabolic signatures associated with each rhodopsin-expressing cell line, demonstrating a highly significant effect of genotype on global metabolite composition (F = 71.679; R