Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

Abstract

Background:The P23H opsin mutant causes the blinding human disease, retinitis pigmentosa. Results: Molecular properties of bovine P23H mutant opsin were characterized in both in vitro and a transgenic C. elegans model. Conclusion: Thermally unstable P23H isorhodopsin containing correct disulfide bond can be slowly regenerated in transgenic C. elegans. Significance: This study produced novel information about the disease-causing P23H mutant opsin.

Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa

“…Their changes could be due to cellular adaptation required to support the biosynthesis of the opsin protein, a G protein-coupled receptor (GPCR). Alternatively, the DE profile of Opsin versus Control cells includes many genes containing cAMP responsive elements (CREs) [52] which could be regulated by the leaky activity of free opsin [53-55] coupled to endogenous G i/o protein signaling [7, 16, 56]. Such genes include but are not limited to Gsta4, Pcx , and Pdk4 involved in metabolic processes; Per1 , Ppargc1a and Egfr1 that regulate gene transcription; the growth factor Prl2c2 ; the neurotransmitter Inhba ; and Ppp1r15a involved in DNA repair (Table 2).…”

“…Thus, it is not surprising that Rho encoding the rod opsin, is the most frequent causal gene among autosomal dominant (ad) RP patients [3, 13]. The P23H mutation, observed in 10% of adRP cases, is a representative Class II mutation that causes opsin misfolding due to its thermal instability [3, 13-16]. Instability of P23H opsin leads to its progressive massive degradation in the rod photoreceptors of P23H knock-in mice [17, 18].…”

“…Even though a valid photoreceptor cell line is unavailable, mammalian cells have been commonly used to study the biosynthesis of rhodopsin and to screen for drug candidates because the pre-ciliary biosynthesis of rhodopsin is generally shared by mammalian cells and rod photoreceptor cells [14, 20, 21]. In mammalian cell cultures, heterologously expressed WT opsin is located on the plasma membrane whereas P23H opsin accumulates in the endoplasmic reticulum (ER) due to its structural instability [14-16, 19-21]. …”

Structural and molecular bases of rod photoreceptor morphogenesis and disease

“…Increasing the levels of rhodopsin mRNA and protein was found to preserve rod cell viability so that as many as four times as many photoreceptor nuclei remained in mice expressing a P23H mutant rhodopsin. This form of rhodopsin is known to fold inefficiently (Liu et al, 1996; Sung et al, 1991), to be unstable (Chen et al, 2014), and to mislocalize to the inner segment (Tam and Moritz, 2006), so it has been assumed to cause cell death through a toxic gain-of-function, such as triggering the unfolded protein response, or toxic stress of the endoplasmic reticulum. The genetic experiments show clearly that the primary mechanism is a dominant negative effect, consistent with the observation that over-expression of wildtype rhodopsin, which has a protective effect on the P23H background, increases, rather than decreases, the percentage of a P23H rhodopsin-EGFP fusion that is transported to the outer segment (Price et al, 2012), and with the observation that P23H rhodopsin has a harmful effect on disk structure in the outer segment (Sakami et al, 2011).…”

High-Throughput Screening Assays to Identify Small Molecules Preventing Photoreceptor Degeneration Caused by the Rhodopsin P23H Mutation

“…To date, there is no effective treatment for this disease, although multiple experimental efforts have been reported [9–17]. Currently, active compounds which showed protective effects in P23H rhodopsin-associated adRP models are limited to two categories: (1) the native chromophore and its analogs with high light sensitivity, low stability, and relatively high toxicity [18–21] and (2) natural antioxidant substances which require high dosages for efficacy and are not suitable for human treatment [12–14]. …”