Rhodopsin Aggregation Research Articles

Aggregation of rhodopsin mutants in mouse models of autosomal dominant retinitis pigmentosa

Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.

Rhodopsin Oligomerization and Aggregation.

Rhodopsin is the light receptor in photoreceptor cells of the retina and a prototypical G protein-coupled receptor. Two types of quaternary structures can be adopted by rhodopsin. If rhodopsin folds and attains a proper tertiary structure, it can then form oligomers and nanodomains within the photoreceptor cell membrane. In contrast, if rhodopsin misfolds, it cannot progress through the biosynthetic pathway and instead will form aggregates that can cause retinal degenerative disease. In this review, emerging views are highlighted on the supramolecular organization of rhodopsin within the membrane of photoreceptor cells and the aggregation of rhodopsin that can lead to retinal degeneration.

Detection of misfolded rhodopsin aggregates in cells by Förster resonance energy transfer.

Rhodopsin is the light receptor in rod photoreceptor cells of the retina that plays a central role in phototransduction and rod photoreceptor cell health. Rhodopsin mutations are the leading known cause of autosomal dominant retinitis pigmentosa, a retinal degenerative disease. A majority of rhodopsin mutations cause misfolding and aggregation of the apoprotein opsin. The nature of aggregates formed by misfolded rhodopsin mutants and the associated cell toxicity is poorly understood. Misfolding rhodopsin 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 rhodopsin aggregation, disease pathogenesis, and evaluate therapeutic strategies. To better understand the aggregation of misfolded rhodopsin mutants, a Förster resonance energy transfer assay has been developed to monitor the aggregation of fluorescently tagged mutant rhodopsins expressed in live cells.

The role of the ER stress-response protein PERK in rhodopsin retinitis pigmentosa.

Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of dominant retinitis pigmentosa (RP), a type of inherited blindness caused by the dysfunction and death of photoreceptor cells. The P23H mutation, the most frequent single cause of RP in the USA, causes rhodopsin misfolding and induction of the unfolded protein response (UPR), an adaptive ER stress response and signalling network that aims to enhance the folding and degradation of misfolded proteins to restore proteostasis. Prolonged UPR activation, and in particular the PERK branch, can reduce protein synthesis and initiate cell death through induction of pro-apoptotic pathways. Here, we investigated the effect of pharmacological PERK inhibition on retinal disease process in the P23H-1 transgenic rat model of retinal degeneration. PERK inhibition with GSK2606414A led to an inhibition of eIF2α phosphorylation, which correlated with reduced ERG function and decreased photoreceptor survival at both high and low doses of PERK inhibitor. Additionally, PERK inhibition increased the incidence of inclusion formation in cultured cells overexpressing P23H rod opsin, and increased rhodopsin aggregation in the P23H-1 rat retina, suggesting enhanced P23H misfolding and aggregation. In contrast, treatment of P23H-1 rats with an inhibitor of eIF2α phosphatase, salubrinal, led to improved photoreceptor survival. Collectively, these data suggest the activation of PERK is part of a protective response to mutant rhodopsin that ultimately limits photoreceptor cell death.

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.

Hsp90 inhibition protects against inherited retinal degeneration

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1−/− cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

Characterization of Membrane Protein Non-native States. 1. Extent of Unfolding and Aggregation of Rhodopsin in the Presence of Chemical Denaturants

Little is known about the general folding mechanisms of helical membrane proteins. Unfolded, i.e., non-native states, in particular, have not yet been characterized in detail. Here, we establish conditions under which denatured states of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied. We investigated the effects of the chemical denaturants sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride (GuHCl), and trifluoroacetic acid (TFA) on rhodopsin's secondary structure and propensity for aggregation. Ellipticity at 222 nm decreases in the presence of maximum concentrations of denaturants in the order TFA > GuHCl > urea > SDS + urea > SDS. Interpretation of these changes in ellipticity in terms of helix loss is challenged because the addition of some denaturants leads to aggregation. Through a combination of SDS-PAGE, dependence of ellipticity on protein concentration, and 1D (1)H NMR we show that aggregates form in the presence of GuHCl, TFA, and urea but not in any concentration of SDS, added over a range of 0.05%-30%. Mixed denaturant conditions consisting of 3% SDS and 8 M urea, added in this order, also did not result in aggregation. We conclude that SDS is able to prevent the exposure of large hydrophobic regions present in membrane proteins which otherwise leads to aggregation. Thus, 30% SDS and 3% SDS + 8 M urea are the denaturing conditions of choice to study maximally unfolded rhodopsin without aggregation.

How does the State of Aggregation of Rhodopsin in Retinal Discs Influence the Variability of its Activated Life Time?

Single photon responses (SPRs) in the retinal rod are less variable than expected assuming that deactivation of the receptor rhodopsin (R∗) occurs in a single memoryless step. It has been suggested that SPRs reproducibility can be explained by a sequential increase of affinity of R∗ to the protein involved in its deactivation arrestin (Arr) which leads to a reduction in the life-time variability of R∗. This increase in affinity is promoted by a serial phosphorylation of rhodopsin catalyzed by rhodopsin- kinase (RK). This deactivation mechanism has been tested successfully by means of stochastic simulation assuming rapid diffusion of all signaling molecules. However, evidence suggests that, in native rod discs, rhodopsin is found forming dimers organized in paracrystalline arrays covering about half of the membrane surface. In this work, we test the hypothesis that packing induced crowding effects, in conjunction with the competitive interactions between R∗ an the other proteins involved in the signaling cascade (G protein (G), RK, and Arr) will influence the variability of the half-time of R∗. In particular, we explore whether the local decrease of inactivated G around R∗ (as it becomes activated by R∗) facilitates interactions of the receptor with Arr and RK, increasing the probability of R∗ deactivation. This would then lead to a reduction in its trial to trial variation. In order to explore these issues, we implement a mesoscopic Monte Carlo simulation in a two-dimensional grid representing the membrane, and follow the stochastic encounters and reactions between the species involved in the signal cascade. We perform the simulations and present data on the variability of the half life of R∗ under two scenarios: rapid diffusion of all proteins, and immobile paracrystalline arrays of rhodopsin.

Rhodopsin in Bovine Rod Outer Segment Disk Membranes Exhibits Two Thermal Transitions

The photoreceptor, rhodopsin is a GPCR in rod outer segment disk membranes. Activation by light converts the dark-adapted form, rhodopsin is to the bleached form, opsin. Differential scanning calorimetry (DSC) studies showed that rhodopsin and opsin each exhibit an irreversible scan rate dependent endothermic transition (Tm) at approximately 72oC and 55oC respectively. We report here a scan rate dependent exothermic transition. Solubilization was used to examine the contribution of the bilayer. Freeze-thaw cycles were used to examine rhodopsin aggregation in these transitions. Disk membranes were sequentially solubilized in octylglucoside (OG) until rhodopsin was completely delipidated. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90°/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. As the OG partitioned into the bilayer the endothermic Tm and Eact (activation energy of denaturation) rapidly decreased. Both then remained constant following rhodopsin solubilization. At low detergent concentration the exothermic Tm increased rapidly then remained constant after solubilization. Unlike the endothermic Eact, the degree of solubilization had little effect on the exothermic transition Eact. Freeze-thaw cycles generated disks in which rhodopsin was aggregated. Increasing aggregation resulted in a decrease in the Tm of the exothermic transition, but had no effect on the endothermic Tm of either rhodopsin or opsin. These results are consistent with earlier cross-linking studies suggesting the exothermic transition results from aggregation. These results indicate that the rhodopsin endothermic transition is due to weakening of the tertiary structure interactions accompanied by changes in the packing of the trans-membrane helices as well as changes in protein-lipid interactions. CD data indicate that rhodopsin secondary structure is almost unchanged.

Effect of Digitonin on the Rhodopsin Meta I‐Meta II Equilibrium¶

ABSTRACTAbsorbance difference spectra were recorded from 10 μs to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20°C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large‐scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time‐resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.

Effect of Digitonin on the Rhodopsin Meta I - Meta II Equilibrium

Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20 degrees C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.

Effect of Digitonin on the Rhodopsin Meta I–Meta II Equilibrium¶

Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20 degrees C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.

Halogenated general anesthetics in visual photoreceptor membranes as examined by static and dynamic properties of rhodopsin.

The visual pigment rhodopsin was used as an intrinsic probe to measure the effect of incorporation of enflurane into bovine rod outer segment disk membranes. At moderately low enflurane concentration, we find that, while extrinsic probes show little membrane perturbation, rhodopsin may experience large changes with respect to the native lipid-protein interactions which modulate some of its properties. Enflurane induces a small blue shift in the pigment lambda max and a strong inhomogeneity in its photochemical behavior. As a function of enflurane concentration, there appears to be an increasing population of rhodopsin molecules for which the metarhodopsin I-->II transition is blocked, while it is accelerated in the remaining population. Under these conditions, electrophoresis and partial membrane solubilization indicate that an increasing amount of rhodopsin does not behave like a free monomeric species. These results are all consistent with a reduction of the membrane order accompanied by lateral aggregation of rhodopsin in the presence of enflurane. By comparison with the perturbation probed by a spin label, the large molecular environment change experienced by the rhodopsin molecule suggests that enflurane may concentrate at the lipid-protein interface.

Functional properties of cattle rhodopsin in soluble complex with phospholipids and deoxycholate.

Abstract— The necessary conditions of bleached rhodopsin to activate GTPase and to regenerate α‐band were studied by changing the number of bound phospholipids to rhodopsin using gel filtration procedure. The number of bound phospholipids per mole of rhodopsin (bPL/rho) in the eluants was reproducibly controlled by the concentration of sodium deoxycholate (DOC) in the elution buffer. The eluants were soluble complexes composed of rhodopsin with original a‐band, disk phospholipids and DOC. The regenerability of α‐band depended on bPL/rho but neither on the concentration of DOC nor on state of aggregation of rhodopsin. The lowest number of bPL/rho for this activity under our experimental conditions was estimated to be30–50 in bPL/rho. GTPase was activated only by such complexes that had a nearly original quantity of bPL/rho in disk membranes. Other complexes with less bPL/rho showed aggregation upon bleaching and did not activate GTPase. The amount of phospholipids present in the disk membranes is sufficient to prevent aggregation of rhodopsin upon bleaching.