Ca2+/recoverin dependent regulation of phosphorylation of the rhodopsin mutant R135L associated with retinitis pigmentosa

No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca2+/recoverin upon phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca2+/recoverin than wild-type rhodopsin. This suggests an involvement of Ca2+/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca2+/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.     

Alterations in the photoactivation pathway of rhodopsin mutants associated with retinitis pigmentosa

The visual photoreceptor rhodopsin undergoes a series of conformational changes upon light activation, eventually leading to the active metarhodopsin II conformation, which is able to bind and activate the G-protein, transducin. We have previously shown that mutant rhodopsins G51V and G89D, associated with retinitis pigmentosa, present photobleaching patterns characterized by the formation of altered photointermediates whose nature remained obscure. Our current detailed UV-visible spectroscopic analysis, together with functional characterization, indicate that these mutations influence the relative stability of the different metarhodopsin photointermediates by altering their equilibria and maintaining the receptor in a nonfunctional light-induced conformation that may be toxic to photoreceptor cells. We propose that G51V and G89D shift the equilibrium from metarhodopsin I towards an intermediate, recently named as metarhodopsin Ib, proposed to interact with transducin without activating it. This may be one of the causes contributing to the molecular mechanisms underlying cell death associated with some retinitis pigmentosa mutations.     

Patterns of retinal light absorption related to retinitis pigmentosa mutants from in silico model structures of rhodopsin

Changes induced by mutations in rhodopsin that are associated with the degenerative visual disease retinitis pigmentosa result in an altered pattern of light absorption according to quantum mechanical simulations and reference experimental works. Eleven single-point mutations associated with retinitis pigmentosa at and in the proximity to the retinal binding pocket of rhodopsin have been modeled in silico and their spectra calculated with the NDOL (Neglect of Differential Overlap accounting L azimuthal quantum number) a priori method. The altered pattern of absorption found would lead to cumulative consequences in energy dissipation with aging. Different energy balances in the case of mutants at the very molecular level, compared to native nonmutated rhodopsin, can cause permanent cellular stress and would play a role in the progression of the retine degenerative process. It could explain the worsening of the pathological condition mostly in adults and suggests the probable beneficial effects of using quenching drugs and protection devices against excess of light in the early stages of life for avoiding or reducing potential damage.     

The diagnostic application of targeted re-sequencing in Korean patients with retinitis pigmentosa

Background

Identification of the causative genes of retinitis pigmentosa (RP) is important for the clinical care of patients with RP. However, a comprehensive genetic study has not been performed in Korean RP patients. Moreover, the genetic heterogeneity found in sensorineural genetic disorders makes identification of pathogenic mutations challenging. Therefore, high throughput genetic testing using massively parallel sequencing is needed.

Results

Sixty-two Korean patients with nonsyndromic RP (46 patients from 18 families and 16 simplex cases) who consented to molecular genetic testing were recruited in this study and targeted exome sequencing was applied on 53 RP-related genes. Causal variants were characterised by selecting exonic and splicing variants, selecting variants with low allele frequency (below 1 %), and discarding the remaining variants with quality below 20. The variants were additionally confirmed by an inheritance pattern and cosegregation test of the families, and the rest of the variants were prioritised using in-silico prediction tools. Finally, causal variants were detected from 10 of 18 familial cases (55.5 %) and 7 of 16 simplex cases (43.7 %) in total. Novel variants were detected in 13 of 20 (65 %) candidate variants. Compound heterozygous variants were found in four of 7 simplex cases.

Conclusion

Panel-based targeted re-sequencing can be used as an effective molecular diagnostic tool for RP.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1723-x) contains supplementary material, which is available to authorized users.     

Cell‐specific differences in the processing of the R14W CAIV mutant associated with retinitis pigmentosa 17

Retinitis pigmentosa is a highly heterogeneous form of inherited blindness which affects more than 1.3 million individuals worldwide. The RP17 form of the disease is caused by an arginine to tryptophan (R14W) mutation in the signal sequence of carbonic anhydrase IV (CAIV). While CAIV is expressed in the choriocapillaries of the eye and renal epithelium, the R14W mutation results in an exclusively ocular phenotype in affected individuals. In order to investigate the mechanism of disease in RP17 and the lack of kidney phenotype, we compared the subcellular localization and post‐translational processing of wild‐type (WT)‐ and mutant‐CAIV in three cell types. We show using immunocytochemistry that unlike WT CAIV which is transported to the plasma membrane of transfected COS‐7 and HT‐1080 cells, the R14W mutant CAIV is retained in the endoplasmic reticulum. Western blot analyses further reveal that whereas the WT CAIV is processed to its mature form in both these cell lines, significant levels of the R14W mutant protein remain in its immature form. Importantly, flow cytometry experiments demonstrate that compared to WT CAIV protein, expression of specifically the R14W CAIV results in an S and G2/M cell‐cycle block, followed by apoptosis. Interestingly, when the above experiments were repeated in the human embryonic kidney cell line, HEK‐293, strikingly different results were obtained. These cells were unaffected by the expression of the R14W mutant CAIV and were able to process the mutant and WT protein equally effectively. This study has important implications for our understanding of the RP17 phenotype. J. Cell. Biochem. 111: 735–741, 2010. © 2010 Wiley‐Liss, Inc.     

Diagnostic challenges in retinitis pigmentosa: genotypic multiplicity and phenotypic variability

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal disorders. Diagnosis can be challenging as more than 40 genes are known to cause non-syndromic RP and phenotypic expression can differ significantly resulting in variations in disease severity, age of onset, rate of progression, and clinical findings. We describe the clinical manifestations of RP, the more commonly known causative gene mutations, and the genotypic-phenotypic correlation of RP.     

Mutation analysis of codons 345 and 347 of rhodopsin gene in Indian retinitis pigmentosa patients

More than 100 mutations have been reported till date in the rhodopsin gene in patients with retinitis pigmentosa. The present study was undertaken to detect the reported rhodopsin gene point mutations in Indian retinitis pigmentosa patients. We looked for presence or absence of codon 345 and 347 mutations in exon 5 of the gene using the technique of allele-specific polymerase chain reaction by designing primers for each mutation. We have examined 100 patients from 76 families irrespective of genetic categories. Surprisingly, in our sample the very widely reported highly frequent mutations of codon 347 (P → S/A/R/Q/L/T) were absent while the codon 345 mutation V → M was seen in three cases in one family (autosomal dominant form) and in one sporadic case (total two families). This is the first report on codon 345 and 347 mutation in Indian retinitis pigmentosa subjects.     

PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor

PAP-1 is an in vitro phosphorylation target of the Pim-1 oncogene. Although PAP-1 binds to Pim-1, it is not a substrate for phosphorylation by Pim-1 in vivo. PAP-1 has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). However, RP9 is a rare disease and only two missense mutations have been described, so the report of a link between PAP-1 and RP9 was tentative. The precise cellular role of PAP-1 was also unknown at that time. We now report that PAP-1 localizes in nuclear speckles containing the splicing factor SC35 and interacts directly with another splicing factor, U2AF35. Furthermore, we used in vitro and in vivo splicing assays to show that PAP-1 has an activity, which alters the pattern of pre-mRNA splicing and that this activity is dependent on the phosphorylation state of PAP-1. We used the same splicing assay to examine the activities of two mutant forms of PAP-1 found in RP9 patients. The results showed that while one of the mutations, H137L, had no effect on splicing activity compared with that of wild-type PAP-1, the other, D170G, resulted in both a defect in splicing activity and a decreased proportion of phosphorylated PAP-1. The D170G mutation may therefore cause RP by altering splicing of retinal genes through a decrease in PAP-1 phosphorylation. These results demonstrate that PAP-1 has a role in pre-mRNA splicing and, given that three other splicing factors have been implicated in adRP, this finding provides compelling further evidence that PAP-1 is indeed the RP9 gene.     

Retinitis pigmentosa-associated rhodopsin mutations in three membrane-located cysteine residues present three different biochemical phenotypes

A large number of mutations in rhodopsin are associated with autosomal dominant retinitis pigmentosa (ADRP). We analyzed the biochemical phenotypes of the ADRP-associated cysteine mutants C167R, C222R, and C264del. C222R behaved as wild type in every aspect testable and is classified as a class I mutant. C167R produced intact protein but did not regenerate with 11-cis retinal and was not transported to the plasma membrane. We confirm its classification as a class IIa mutant. C264del represents a novel phenotype, which we propose to call class III. It produced a truncated protein of 27kDa that failed to regenerate with 11-cis retinal and was not targeted to the plasma membrane.     

Misfolded rhodopsin mutants display variable aggregation properties

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.     

Crystal structure of the C-terminal domain of splicing factor Prp8 carrying retinitis pigmentosa mutants

Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an alpha/beta structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn(2+) coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn(2+)-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.     

Screening for homozygosity by descent in families with autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous disease and an important cause of blindness in the state of Andhra Pradesh in India. In an attempt to identify the disease locus in families with the recessive form of the disease, we used the approach of screening for homozygosity by descent in offspring of consanguineous and nonconsanguineous families with RP. Microsatellite markers closely flanking 21 known candidate genes for RP were genotyped in parents and affected offspring to determine whether there was homozygosity at these loci that was shared by affected individuals of a family. This screening approach may be a rapid preliminary method to test known loci for possible cosegregation with disease.     

Molecular mechanisms of disease for mutations at Gly-90 in rhodopsin

Two different mutations at Gly-90 in the second transmembrane helix of the photoreceptor protein rhodopsin have been proposed to lead to different phenotypes. G90D has been classically associated with congenital night blindness, whereas the newly reported G90V substitution was linked to a retinitis pigmentosa phenotype. Here, we used Val/Asp replacements of the native Gly at position 90 to unravel the structure/function divergences caused by these mutations and the potential molecular mechanisms of inherited retinal disease. The G90V and G90D mutants have a similar conformation around the Schiff base linkage region in the dark state and same regeneration kinetics with 11-cis-retinal, but G90V has dramatically reduced thermal stability when compared with the G90D mutant rhodopsin. The G90V mutant also shows, like G90D, an altered photobleaching pattern and capacity to activate Gt in the opsin state. Furthermore, the regeneration of the G90V mutant with 9-cis-retinal was improved, achieving the same A(280)/A(500) as wild type isorhodopsin. Hydroxylamine resistance was also recovered, indicating a compact structure around the Schiff base linkage, and the thermal stability was substantially improved when compared with the 11-cis-regenerated mutant. These results support the role of thermal instability and/or abnormal photoproduct formation in eliciting a retinitis pigmentosa phenotype. The improved stability and more compact structure of the G90V mutant when it was regenerated with 9-cis-retinal brings about the possibility that this isomer or other modified retinoid analogues might be used in potential treatment strategies for mutants showing the same structural features.     

Identification of novel rhodopsin mutations associated with retinitis pigmentosa by GC-clamped denaturing gradient gel electrophoresis.

Retinitis pigmentosa (RP) is a group of disorders characterized by progressive degeneration of the outer retina, resulting in night blindness, visual field loss, an abnormal electroretinogram, and characteristic retinal pigmentary changes. An important step in the understanding of RP has been the recognition that some cases of autosomal dominant RP (ADRP) are caused by mutations in the rhodopsin gene. Multiple different point mutations within the coding sequence of the rhodopsin gene have been associated with ADRP. We have developed a GC-clamped denaturing-gradient-gel electrophoresis (DGGE) assay for the coding region of the rhodopsin gene and have used this assay to screen ADRP patients for mutations. The assay consists of amplifying with PCR the five exons of the rhodopsin gene and then analyzing each PCR product by DGGE. We have used this assay to detect three previously unreported rhodopsin base substitutions associated with ADRP. The use of this assay to identify ADRP patients who have various rhodopsin mutations has allowed us to begin studies seeking to correlate molecular genotype with clinical phenotype. Furthermore, GC-clamped DGGE has allowed us to identify families with ADRP not caused by a rhodopsin mutation. Such families will be important in the search for other genes involved in ADRP.     

Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138^3.53 and the multiple mutant containing V227^5.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138^3.53, V227^5.62, V250^6.33, V254^6.37 and I255^6.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.     

3D modeling of the activated states of constitutively active mutants of rhodopsin

The activated (R*) states in constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) are presumably characterized by lower energies than the resting (R) states. If specific configurations of TM helices differing by rotations along the long transmembrane axes possess energies lower than that in the R state for pronounced CAMs, but not for non-CAMs, these particular configurations of TM helices are candidate 3D models for the R* state. The hypothesis was studied in the case of rhodopsin, the only GPCR for which experimentally determined 3D models of the R and R* states are currently available. Indeed, relative energies of the R* state were significantly lower than that of the R state for the rhodopsin mutants G90D/M257Y and E113Q/M257Y (strong CAMs), but not for G90D, E113Q, and M257Y (not CAMs). Next, the developed build-up procedure successfully identified few similar configurations of the TM helical bundle of G90D/M257Y and E113Q/M257Y as possible candidates for the 3D model of the R* state of rhodopsin, all of them being in good agreement with the model suggested by experiment. Since constitutively active mutants are known for many of GPCRs belonging to the large rhodopsin-like family, this approach provides a way for predicting possible 3D structures corresponding to the activated states of the TM regions of many GPCRs for which CAMs have been identified.