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A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation
摘要： Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS. Two stabilization assays of P23H mutant opsin were developed and validated, one based on b-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identi?ed changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS. Our ?ndings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for dif?cult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.
关键词： ocular pharmacology,retinal degeneration,GPCR,phototransduction,rod photoreceptors,rhodopsin,misfolded protein
更新于2025-11-21 11:20:48
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Molecular interactions and mutational impact upon rhodopsin (G90→D90) for hindering dark adaptation of eye: A comparative structural level outlook for signaling mechanism in night blindness
摘要： For night blindness, a detailed structural exploration of the interactions among G-protein receptor rhodopsin, transducin and arrestin was performed. Rhodopsin is responsible for dim light vision while a point mutation (G90→D90) results in an adverse change in its photo-transduction. The validated 3D models of the three proteins were utilized, and upon mutation and interactions, rhodopsin attained higher stability (evaluated through thermodynamic energy calculations, electrostatic surface potential and solvent accessible area), thereby participating strongly with transducin. Conformational switches in mutated rhodopsin also depicted a firm conformation with few 310 helices accompanied by increased percentage of pure α-helices and sheets. All evaluations were corroborated through paired T-tests. Glu33 (glycosylated unit in the N-terminal zone) of rhodopsin plays a chief role in the overall interaction pattern. Arg69 and Glu33 from wild-type rhodopsin participated in ionic interactions, while the latter set of ionic interaction remained preserved even after mutation. Cys323 (C-terminal residue) and Arg69 formed H-bonds from the wild-type rhodopsin. Cys323 exceptionally supports cellular signaling pattern in the non-mutated situation and for the non-sufferers of night-blindness. Ser297 and Tyr43 from mutated rhodopsin reside in helices and interact with Thr32 of transducin, preserving the steady conformation in activated interacted state, even in the dark. Ser297 lies adjoined to Lys296 (retinal attachment site), which resides in NPXXY motif (an 'activation switch' for signal transduction). Thus, the molecular facet for involvement of photo-transduction, which holds a paramount zone in ophthalmology, was dealt with. This might instigate the future prospect for drug discovery to prevent such mutations.
关键词： Rhodopsin,Thermodynamic Stabilities,Conformational Fluctuations,Mutational Impact
更新于2025-09-23 15:23:52
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Structural and Functional Consequences of the Weak Binding of Chlorin e6 to Bovine Rhodopsin
摘要： The chlorophyll-derivative chlorin e6 (Ce6) identified in the retinas of deep-sea ocean fish is proposed to play a functional role in red bioluminescence detection. Fluorescence and 1H NMR spectroscopy studies with the bovine dim-light photoreceptor, rhodopsin, indicate that Ce6 weakly binds to it with μM affinity. Absorbance spectra prove that red light sensitivity enhancement is not brought about by a shift in the absorbance maximum of rhodopsin. 19F NMR experiments with samples where 19F labels are either placed at the cytoplasmic binding site or incorporated as fluorinated retinal, indicate that the cytoplasmic domain is highly perturbed by binding, while little to no changes are detected near the retinal. Binding of Ce6 also inhibits G protein activation. Chemical shift changes in 1H,15N NMR spectroscopy of 15N-Trp labeled bovine rhodopsin reveal that Ce6 binding perturbs the entire structure. These results provide experimental evidence that Ce6 is an allosteric modulator of rhodopsin.
关键词： G protein coupled receptor,chlorophyll-derivative,porphyrin,night vision,light activation,photosensitization,bovine rhodopsin,Allosteric modulator
更新于2025-09-23 15:22:29
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Role of Rhodopsins as Circadian Photoreceptors in the Drosophila melanogaster
摘要： Light profoundly affects the circadian clock and the activity levels of animals. Along with the systematic changes in intensity and spectral composition, over the 24-h day, light shows considerable irregular fluctuations (noise). Using light as the Zeitgeber for the circadian clock is, therefore, a complex task and this might explain why animals utilize multiple photoreceptors to entrain their circadian clock. The fruit fly Drosophila melanogaster possesses light-sensitive Cryptochrome and seven Rhodopsins that all contribute to light detection. We review the role of Rhodopsins in circadian entrainment, and of direct light-effects on the activity, with a special emphasis on the newly discovered Rhodopsin 7 (Rh7). We present evidence that Rhodopsin 6 in receptor cells 8 of the compound eyes, as well as in the extra retinal Hofbauer-Buchner eyelets, plays a major role in entraining the fly’s circadian clock with an appropriate phase-to-light–dark cycles. We discuss recent contradictory findings regarding Rhodopsin 7 and report original data that support its role in the compound eyes and in the brain. While Rhodopsin 7 in the brain appears to have a minor role in entrainment, in the compound eyes it seems crucial for fine-tuning light sensitivity to prevent overshooting responses to bright light.
关键词： electroretinogram,Rhodopsins,entrainment,retina,Rhodopsin 7,immunocytochemistry
更新于2025-09-23 15:22:29
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Clinically Relevant Outcome Measures for the I307N Rhodopsin Mouse: A Model of Inducible Autosomal Dominant Retinitis Pigmentosa
摘要： PURPOSE. The I307N rhodopsin (Rho) mouse is a light-inducible model of autosomal dominant retinitis pigmentosa (adRP) that may be useful in testing therapies. We investigated the time-course of retinal changes of the I307N Rho mouse with spectral-domain optical coherence tomography (SD-OCT). METHODS. SD-OCT was performed up to day 30 after light damage; electroretinography (ERG) was employed to evaluate photoreceptor function. We utilized ImageJ to analyze reflectivity of the retina. We used light and electron microscopy to assess retinal organization. We stained synaptophysin and zonula occludins-1 with immunohistochemistry to determine injury to the plexiform layers and retinal pigment epithelium (RPE). We performed lectin staining to evaluate retinal blood vessels. RESULTS. Retinal degeneration increased with longer exposures to light. An increase in retinal thickness was detected by SD-OCT on day 1 after light challenge followed by loss of the outer nuclear layer (ONL) by day 8. Degeneration was most severe in the nasal and inferior retina. Hyper-reflectivity on SD-OCT developed as early as 1 day after light exposure. Disorganization of the ONL, condensation of photoreceptor chromatin, disruption of the outer limiting membrane, and disarray of outer segments were associated with the hyper-reflectivity. Retraction of the outer plexiform synapses and resorption of the subretinal detachment contributed to retinal thinning. The RPE remained intact, whereas atrophied major retinal vessels were evident after light damage. CONCLUSIONS. Our time-course analysis of retinal degeneration in the I307N Rho mouse with SD-OCT and other outcome measures should enable the use of the mouse model in preclinical efficacy studies and mechanistic studies.
关键词： SD-OCT,rhodopsin,electroretinography,retinitis pigmentosa,hyper-reflectivity
更新于2025-09-23 15:21:01
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Maintenance of Rhodopsin levels in <i>Drosophila</i> photoreceptor and phototransduction requires Protein Kinase D
摘要： During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. In this study, we report that Protein kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKDH), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKDH. Our results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.
关键词： Electroretinogram (ERG),Phototransduction,Retinal degeneration,Rhodopsin,Drosophila,Rhodopsin loaded vesicle (RLVs),Protein Kinase D
更新于2025-09-19 17:15:36
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Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin
摘要： Mastigocladopsis repens halorhodopsin (MrHR) is a Cl?-pumping rhodopsin that belongs to a distinct cluster far from other Cl? pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I? but cannot transport it. I?-bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl?-pumping photocycle, Cl? moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I?-bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl?-binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl?-binding affinity nor the absorption spectrum, but completely eliminated the Cl?-pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H? release was detected for photolyzed MrHR by using an indium-tin oxide electrode, which acts as a good time-resolved pH sensor. This H? release disappeared in the I?-bound form and in the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl? movement to the CP channel.
关键词： Flash photolysis,Microbial rhodopsin,Photocycle,Halorhodopsin,Light-driven chloride pump
更新于2025-09-19 17:15:36
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Optogenetic modulation of cortical neurons using Organic Light Emitting Diodes (OLEDs)
摘要： Approach - Cultured mouse cortical neurons were transfected with light-sensitive opsins such as blue-light sensitive channel-rhodopsin (ChR2) and green-light sensitive chimeric channel-rhodopsin (C1V1tt) and stimulated using blue and green OLEDs (with 455 and 520 nm peak emission spectra respectively) at a power of 1 mW/mm2 under pulsed conditions. vivo in a transgenic mouse model. Significance - The above results indicate the feasibility of generating sufficient luminance from OLEDs to perform neuromodulation both in vitro and in vivo. This opens up the possibility of developing thin, flexible OLED films with multiple stimulation sites that can conform to the shape of the neuronal targets in the brain or the peripheral nervous system. However, stability of these OLEDs under chronic conditions still needs to be carefully assessed with appropriate packaging approaches. Main results - We demonstrate neuromodulation and optostimulus-locked, single unit-neuronal activity in neurons expressing stimulating opsins (34 units on n=4 MEAs, each with 16 recordable channels). We also validated the optostimulus-locked response in preliminary experiments in a channel-rhodopsin expressing transgenic mouse model, where at least three isolatable single neuronal cortical units respond to OLED stimulation.
关键词： C1V1tt,Neuromodulation,Channel-rhodopsin,OLEDs,Optogenetics
更新于2025-09-19 17:13:59
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Spectral tuning mediated by helix III in butterfly long wavelength-sensitive visual opsins revealed by heterologous action spectroscopy
摘要： Absorption spectra of opsin-based pigments are tuned from the UV to the red regions by interactions of the chromophore with surrounding amino acid residues. Both vertebrates and invertebrates possess long-wavelength-sensitive (LWS) opsins, which underlie color vision involving “red” sensing. The LWS opsins have independently evolved in each lineage, which suggests the existence of diverse mechanisms in spectral tuning. In vertebrate LWS opsins, the mechanisms underlying spectral tuning have been well characterized by spectroscopic analyses with recombinant pigments of wild type (WT) and mutant opsins. However in invertebrate LWS opsins including insect ones, the mechanisms are largely unknown due to the difficulty in obtaining recombinant pigments. Here we have overcome the problem by analyzing heterologous action spectra based on light-dependent changes in the second messenger in opsin-expressing cultured cells. We found that WTs of two LWS opsins of the butterfly, Papilio xuthus, PxRh3 and PxRh1 have the wavelengths of the absorption maxima at around 570 nm and 540 nm, respectively. Analysis of a series of chimeric mutants showed that helix III is crucial to generating a difference of about 15 nm in the wavelength of absorption maxima of these LWS opsins. Further site-directed mutations in helix III revealed that amino acid residues at position 116 and 120 (bovine rhodopsin numbering system) are involved in the spectral tuning of PxRh1 and PxRh3, suggesting a different spectral tuning mechanism from that of primate LWS opsins.
关键词： Red sensitivity,Rhodopsin,Color vision,Convergent evolution
更新于2025-09-19 17:13:59
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Revealing Conformational Transitions in G-Protein Coupled Receptor Rhodopsin upon Phosphorylation
摘要： GPCRs have evolved as highly specialized cellular machinery that can dictate biological outcomes in response to diverse stimuli. Specifically, they induce multiple pathway responses upon structural perturbations induced at local protein sites. GPCRs utilize concurrent strategy involving a central transmembrane topology and biochemical modifications for precise functional implementation. However, the specific role of latter is not decomposed due to the lack of precise probing techniques that can characterize receptor dynamics upon biochemical modifications. Phosphorylation is known to be one of the critical biochemical modifications in GPCRs that aids in receptor desensitization via arrestin binding. Here, we carried out all-atom molecular dynamics (MD) simulations of rhodopsin in membrane environment to study its conformational dynamics induced upon phosphorylation. Interestingly, our comparative analysis of non-phosphorylated and phosphorylated rhodopsin structure demonstrated enhanced receptor stability upon phosphorylation at the C-terminal region that leads to the opening of the extracellular part of the transmembrane helices. In addition, monitoring of the distinct number of phosphorylation states showed that less number of phosphorylated residues does not bring about appropriate conformational changes in the extracellular region. Since phosphorylation results in receptor desensitization and recycling of ligand, our findings provide significant insights into the conformational dynamics of the mechanism of ligand exit from the receptor.
关键词： conformational dynamics,molecular dynamics simulations,GPCRs,phosphorylation,rhodopsin
更新于2025-09-12 10:27:22