C Grayson

Unfolding retinal dystrophies: a role for molecular chaperones?

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.

An atypical phenotype of macular and peripapillary retinal atrophy caused by a mutation in the RP2 gene

Aims: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss. Methods: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband’s lymphoblastoid cells were examined for protein expression. Results: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected. Conclusion: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.

Organization on the plasma membrane of the retinitis pigmentosa protein RP2: investigation of association with detergent-resistant membranes and polarized sorting.

Mutations in the retinitis pigmentosa protein gene RP2 account for up to 15% of X-linked retinitis pigmentosa. RP2 is a novel protein of unknown function, which is targeted to the plasma membrane by dual N-terminal acyl-modification. Dual-acylated proteins are targeted to lipid rafts, and some are subject to polarized sorting. Therefore we investigated the organization of RP2 on the plasma membrane. Endogenous RP2 protein was predominantly localized at the plasma membrane, and exogenously expressed green-fluorescent-protein-tagged protein was also targeted to the membrane in a wide range of cultured cells. High levels of endogenous RP2 protein were present in HeLa cells and in the retinal pigment epithelium-derived cell line ARPE19. A significant proportion of RP2 in cultured neuroblastoma cells was associated with detergent-resistant membranes (DRMs), but much less than other dually acylated proteins (e.g. Lyn and Fyn). In contrast, the RP2-interacting protein Arl3 (ADP-ribosylation factor-like 3) was not found to be associated with DRMs. The association of RP2 with DRMs was cholesterol-dependent. In polarized epithelial cells in culture and in vivo, RP2 was present in both the apical and basolateral domains of the plasma membrane. These data show that RP2 is not specific to either domain, unlike some other dually acylated proteins. Interestingly, the level of RP2 protein increased in the epithelial cell line Caco-2 with differentiation and polarization. These data show that RP2 is present on the membrane of all cell types examined both in vitro and in vivo, and that RP2 associates with lipid rafts, suggesting a potential role for the protein in signal transduction.

Assay and functional analysis of the ARL3 effector RP2 involved in X-linked retinitis pigmentosa

Mutations in RP2 cause X-linked retinitis pigmentosa and also macular and peripapillary atrophy. RP2 is a functional homologue of the tubulin folding cofactor, cofactor C, as it can replace the beta tubulin GTPase stimulating activity of cofactor C in an in vitro assay. An important difference between RP2 and cofactor C is their subcellular localization. RP2 is targeted to the cytoplasmic face of the plasma membrane by dual N-terminal acylation, and this post-translational modification is important for protein function. The activity of tubulin folding cofactors is modulated by certain ADP ribosylation factor-like (Arl) proteins. It has been shown that RP2 can interact directly with Arl3. Here we describe the methodologies that we have developed to analyze the interaction of RP2 with Arl3 and to investigate the effect of RP2 post-translational modifications on its subcellular and tissue localization.