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; R2?=?0.93724; p?=?0.001). Pairwise comparisons and background-subtracted analyses identified consistent alterations in arginine and proline metabolism, glutathione metabolism, and the TCA cycle, nucleotide, amino acid metabolism, redox regulation, and mitochondrial function in cells expressing misfolded P23A. Pathway enrichment highlighted key metabolites in the respective pathways as candidate biomarkers for the rhodopsin P23A mutation. As this study employs a non-retinal cell system, the observed metabolic changes reflect conserved responses to rhodopsin misfolding and proteostatic stress in the ER rather than a direct model of rod cell degeneration. Our findings support the hypothesis that there is a biochemical link, most likely the UPR, between rhodopsin folding/misfolding status and metabolic homeostasis and suggest that targeted metabolic modulation may offer a complementary therapeutic avenue for treating RP.