retinaldehyde and Eye-Diseases--Hereditary

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. Of these apparent class II variants, 67 exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts relative to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest that many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two of these retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

Topics: Diterpenes; Drug Resistance; Eye Diseases, Hereditary; HEK293 Cells; Humans; Mutation; Retinaldehyde; Rhodopsin

We use molecular dynamics (MD) simulations to determine the binding properties of different retinoid species to cellular retinaldehyde binding protein (CRALBP). The complexes formed by 9-cis-retinal or 11-cis-retinal bound to both the native protein and the R234W mutant, associated to Bothnia-retina dystrophy, are investigated. The presented studies are also complemented by analysis of the binding structures of the CRALBP/9-cis-retinol and CRALBP/9,13-dicis-retinal complexes. We find that the poor X-ray scattering properties of the polyene tail of the ligand in all wild-type complexes can be attributed to a high mobility of this region, which does not localize in a single binding conformation even at very low temperatures. Our simulations report a clear difference in the residual solvation pattern in CRALBP complexes with either 9-cis- or 9,13-dicis-retinal. The reported structures indicate that the microsolvation properties of the ligand are the key structural element triggering the very recently discovered isomerase activity of this protein. The binding geometries obtained by MD simulations are validated by calculation of the respective optical spectra by the ZINDO/S semiempirical method, which can reproduce with good qualitative agreement the different red-shifts of the first absorption band of the different complexes.

Topics: Carrier Proteins; Diterpenes; Eye Diseases, Hereditary; Humans; Molecular Dynamics Simulation; Mutation; Protein Conformation; Retinal Diseases; Retinaldehyde

To evaluate phenotypes caused by different RLBP1 mutations in autosomal recessive retinitis pigmentosa of Bothnia type.. Compound heterozygotes for mutations in the RLBP1 gene [c.677T>A]+[c.700C>T] (p.M226K+p.R234W), n = 10, aged 7-84 years, and homozygotes c.677T>A (p.M226K), n = 2, aged 63 and 73 years, were studied using visual acuity (VA), low-contrast VA, visual fields (VFs) and optical coherence tomography (OCT). Retrospective VA and VFs, standardized dark adaptation and full-field electroretinograms (ERGs) were analysed and prolonged dark adaptometry and ERG (at 24 hr) were performed.. Progressive decline of VA and VF areas was age-dependent. Retinal degenerative maculopathy, peripheral degenerative changes and retinitis punctata albescens (RPA) were present. Early retinal thinning in the central foveal, foveal (Ø 1 mm), and inner ring (Ø 3 mm) in the macular region, with homogenous, high-reflectance RPA changes, was visualized in and adjacent to the retinal pigment epithelium/choriocapillaris using OCT. Reduced dark adaptation and affected ERGs were present in all ages. Prolonged dark adaptation and ERG (at 24 hr), an increase in final threshold, and ERG rod and mixed rod/cone responses were found.. The two RLBP1 genotypes presented a phenotypical and electrophysiological expression of progressive retinal disease similar to that previously described in homozygotes for the c.700C>T (p.R234W) RLBP1 mutation. The uniform phenotypical expression of RLBP1 mutations is relevant information for the disease and of importance in planning future treatment strategies.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carrier Proteins; Child; Databases, Genetic; DNA Mutational Analysis; Electroretinography; Eye Diseases, Hereditary; Female; Fluorescein Angiography; Fundus Oculi; Genetic Association Studies; Humans; Male; Middle Aged; Mutation; Phenotype; Retinal Diseases; Retinaldehyde; Retrospective Studies; Tomography, Optical Coherence; Young Adult

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.

Topics: Amino Acid Substitution; Animals; Cattle; Cell Line, Tumor; COS Cells; Diterpenes; Eye Diseases, Hereditary; Genetic Diseases, X-Linked; Humans; Mutation, Missense; Myopia; Night Blindness; Protein Stability; Protein Structure, Tertiary; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship