Unraveling the mystery of ocular retinoid turnover: Insights from albino mice and the role of STRA6.

A delicate balance between photon absorption for vision and the protection of photoreceptors from light damage is pivotal for ocular health. This equilibrium is governed by the light-absorbing 11-cis-retinylidene chromophore of visual pigments, which, upon bleaching, transforms into all-trans-retinal and undergoes regeneration through an enzymatic pathway, named the visual cycle. Chemical side reactions of retinaldehyde during the recycling process can generate by-products that may result in a depletion of retinoids. In our study, we have clarified the crucial roles played by melanin pigmentation and the retinoid transporter STRA6 in preventing this loss and preserving the integrity of the visual cycle. Our experiments initially confirmed that consecutive green and blue light bleaching of isolated bovine rhodopsin produced 9-cis and 13-cis retinal. The same unusual retinoids were found in the retinas of mice exposed to intense light, with elevated concentrations observed in albino mice. Examining the metabolic fate of these visual cycle byproducts revealed that 9-cis-retinal, but not 13-cis-retinal, was recycled back to all-trans-retinal through an intermediate called isorhodopsin. However, investigations in Stra6 knockout mice unveiled that the generation of these visual cycle byproducts correlated with a light-induced loss of ocular retinoids and visual impairment. Collectively, our findings uncover important novel aspects of visual cycle dynamics, with implications for ocular health and photoreceptor integrity.

Genetic deletion of Bco2 and Isx establishes a golden mouse model for carotenoid research.

OBJECTIVE: Low plasma levels of carotenoids are associated with mortality and chronic disease states. Genetic studies in animals revealed that the tissue accumulation of these dietary pigments is associated with the genes encoding ß-carotene oxygenase 2 (BCO2) and the scavenger receptor class B type 1 (SR-B1). Here we examined in mice how BCO2 and SR-B1 affect the metabolism of the model carotenoid zeaxanthin that serves as a macular pigment in the human retina. METHODS: We used mice with a lacZ reporter gene knock-in to determine Bco2 expression patterns in the small intestine. By genetic dissection, we studied the contribution of BCO2 and SR-B1 to zeaxanthin uptake homeostasis and tissue accumulation under different supply conditions (50 mg/kg and 250 mg/kg). We determined the metabolic profiles of zeaxanthin and its metabolites in different tissues by LC-MS using standard and chiral columns. An albino Isx-/-/Bco2-/- mouse homozygous for Tyrc-2J was generated to study the effect of light on ocular zeaxanthin metabolites. RESULTS: We demonstrate that BCO2 is highly expressed in enterocytes of the small intestine. Genetic deletion of Bco2 led to enhanced accumulation of zeaxanthin, indicating that the enzyme serves as a gatekeeper of zeaxanthin bioavailability. Relaxing the regulation of SR-B1 expression in enterocytes by genetic deletion of the transcription factor ISX further enhanced zeaxanthin accumulation in tissues. We observed that the absorption of zeaxanthin was dose-dependent and identified the jejunum as the major zeaxanthin-absorbing intestinal region. We further showed that zeaxanthin underwent oxidation to ε,ε-3,3'-carotene-dione in mouse tissues. We detected all three enantiomers of the zeaxanthin oxidation product whereas the parent zeaxanthin only existed as (3R, 3'R)-enantiomer in the diet. The ratio of oxidized to parent zeaxanthin varied between tissues and was dependent on the supplementation dose. We further showed in an albino Isx-/-/Bco2-/- mouse that supra-physiological supplementation doses (250 mg/kg) with zeaxanthin rapidly induced hypercarotenemia with a golden skin phenotype and that light stress increased the concentration of oxidized zeaxanthin in the eyes. CONCLUSIONS: We established the biochemical basis of zeaxanthin metabolism in mice and showed that tissue factors and abiotic stress affect the metabolism and homeostasis of this dietary lipid.

ASTER-B regulates mitochondrial carotenoid transport and homeostasis.

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by ß-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon ß-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by ß-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-ß-cyclodextrin treatment enhanced zeaxanthin but not ß-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.

Genetic tuning of ß-carotene oxygenase-1 activity rescues cone photoreceptor function in STRA6-deficient mice.

Rod and cone photoreceptors in the retina mediate dim light and daylight vision, respectively. Despite their distinctive functions, rod and cone visual pigments utilize the same vitamin A-derived chromophore. To sustain vision, vitamin A precursors must be acquired in the gut, metabolized, and distributed to the eyes. Deficiencies in this pathway in inherited ocular disease states deplete cone photoreceptors from chromophore and eventually lead to cell death, whereas the more abundant rod photoreceptors are less affected. However, pathways that support cone function and survival under such conditions are largely unknown. Using biochemical, histological, and physiological approaches, we herein show that intervention with ß-carotene in STRA6-deficient mice improved chromophore supply to cone photoreceptors. Relieving the inherent negative feedback regulation of ß-carotene oxygenase-1 activity in the intestine by genetic means further bolstered cone photoreceptor functioning in the STRA6-deficient eyes. A vitamin A-rich diet, however, did not improve cone photoreceptor function in STRA6-deficiency. We provide evidence that the beneficial effect of ß-carotene on cones results from favorable serum kinetics of retinyl esters in lipoproteins. The respective alterations in lipoprotein metabolism maintained a steady supply of retinoids to the STRA6-deficient eyes, which ameliorated the competition for chromophore between rod and cone photoreceptors. Together, our study elucidates a cone photoreceptor-survival pathway and unravels an unexpected metabolic connection between the gut and the retina.

Diabetes Aggravates Photoreceptor Pathologies in a Mouse Model for Ocular Vitamin A Deficiency.

Emerging evidence indicates that diabetes disturbs photoreceptor function and vitamin A homeostasis. However, the biochemical basis of this phenotype is not well established. Here, we compared the effects of streptozotocin-induced diabetes in wild-type (WT) mice and Stra6-/- mice, a mouse model for ocular vitamin A deficiency. After 8 weeks, diabetes increased serum retinyl esters in mice of both genotypes. The eyes of diabetic WT mice displayed increased superoxide levels but no changes in retinoid concentrations. Diabetic Stra6-/- mice showed increased ocular retinoid concentrations, but superoxide levels remained unchanged. After 30 weeks, significant alterations in liver and fat retinoid concentrations were observed in diabetic mice. Diabetic WT mice exhibited a decreased expression of visual cycle proteins and a thinning of the photoreceptor layer. Stra6-/- mice displayed significantly lower ocular retinoid concentration than WT mice. An altered retinal morphology and a reduced expression of photoreceptor marker genes paralleled these biochemical changes and were more pronounced in the diabetic animals. Taken together, we observed that diabetes altered vitamin A homeostasis in several organ systems and aggravated photoreceptor pathologies in the vitamin-deficient mouse eyes.

Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A.

Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. Here, we used a genetic dissection approach in mice to examine the contributions of these proteins in select tissues. We observed that ISX deficiency increased utilization of both preformed and provitamin A. We found that increased storage of retinoids in peripheral tissues of ISX-deficient mice was dependent on STRA6 and induced by retinoid signaling. In addition, double-mutant mice exhibited a partial rescue of the Stra6 mutant ocular phenotype. This rescue came at the expense of a massive accumulation of vitamin A in other tissues, demonstrating that vitamin A is randomly distributed when present in excessive amounts. Remarkably, provitamin A supplementation of mutant mice induced the expression of the RBP receptor 2 in the liver and was accompanied by increased hepatic retinyl ester stores. Taken together, these findings indicate dynamic crosstalk between the delivery pathways for this essential nutrient and suggest that hepatic reuptake of vitamin A takes place when excessive amounts circulate in the blood.

Aster proteins mediate carotenoid transport in mammalian cells.

Some mammalian tissues uniquely concentrate carotenoids, but the underlying biochemical mechanism for this accumulation has not been fully elucidated. For instance, the central retina of the primate eyes displays high levels of the carotenoids, lutein, and zeaxanthin, whereas the pigments are largely absent in rodent retinas. We previously identified the scavenger receptor class B type 1 and the enzyme ß-carotene-oxygenase-2 (BCO2) as key components that determine carotenoid concentration in tissues. We now provide evidence that Aster (GRAM-domain-containing) proteins, recently recognized for their role in nonvesicular cholesterol transport, engage in carotenoid metabolism. Our analyses revealed that the StART-like lipid binding domain of Aster proteins can accommodate the bulky pigments and bind them with high affinity. We further showed that carotenoids and cholesterol compete for the same binding site. We established a bacterial test system to demonstrate that the StART-like domains of mouse and human Aster proteins can extract carotenoids from biological membranes. Mice deficient for the carotenoid catabolizing enzyme BCO2 concentrated carotenoids in Aster-B protein-expressing tissues such as the adrenal glands. Remarkably, Aster-B was expressed in the human but not in the mouse retina. Within the retina, Aster-B and BCO2 showed opposite expression patterns in central versus peripheral parts. Together, our study unravels the biochemical basis for intracellular carotenoid transport and implicates Aster-B in the pathway for macula pigment concentration in the human retina.

The vitamin A transporter STRA6 adjusts the stoichiometry of chromophore and opsins in visual pigment synthesis and recycling.

The retinal pigment epithelium of the vertebrate eyes acquires vitamin A from circulating retinol binding protein for chromophore biosynthesis. The chromophore covalently links with an opsin protein in the adjacent photoreceptors of the retina to form the bipartite visual pigment complexes. We here analyzed visual pigment biosynthesis in mice deficient for the retinol-binding protein receptor STRA6. We observed that chromophore content was decreased throughout the life cycle of these animals, indicating that lipoprotein-dependent delivery pathways for the vitamin cannot substitute for STRA6. Changes in the expression of photoreceptor marker genes, including a downregulation of the genes encoding rod and cone opsins, paralleled the decrease in ocular retinoid concentration in STRA6-deficient mice. Despite this adaptation, cone photoreceptors displayed absent or mislocalized opsins at all ages examined. Rod photoreceptors entrapped the available chromophore but exhibited significant amounts of chromophore-free opsins in the dark-adapted stage. Treatment of mice with pharmacological doses of vitamin A ameliorated the rod phenotype but did not restore visual pigment synthesis in cone photoreceptors of STRA6-deficient mice. The imbalance between chromophore and opsin concentrations of rod and cone photoreceptors was associated with an unfavorable retinal physiology, including diminished electrical responses of photoreceptors to light, and retinal degeneration during aging. Together, our study demonstrates that STRA6 is critical to adjust the stoichiometry of chromophore and opsins in rod and cone photoreceptors and to prevent pathologies associated with ocular vitamin A deprivation.

Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina.

The RLBP1 gene encodes the 36 kDa cellular retinaldehyde-binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Müller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a-mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis from ß-carotene.

There is increasing recognition that dietary lipids can affect the expression of genes encoding their metabolizing enzymes, transporters, and binding proteins. This mechanism plays a pivotal role in controlling tissue homeostasis of these compounds and avoiding diseases. The regulation of retinoid biosynthesis from ß-carotene (BC) is a classic example for such an interaction. The intestine-specific homeodomain transcription factor (ISX) controls the activity of the vitamin A-forming enzyme ß-carotene oxygenase-1 in intestinal enterocytes in response to increasing concentration of the vitamin A metabolite retinoic acid. However, it is unclear how cells control the concentration of the signaling molecule in this negative-feedback loop. We demonstrate in mice that the sequestration of retinyl esters by the enzyme lecithin:retinol acyltransferase (LRAT) is central for this process. Using genetic and pharmacological approaches in mice, we observed that in LRAT deficiency, the transcription factor ISX became hypersensitive to dietary vitamin A and suppressed retinoid biosynthesis. The dysregulation of the pathway resulted in BC accumulation and vitamin A deficiency of extrahepatic tissues. Pharmacological inhibition of retinoid signaling and genetic depletion of the Isx gene restored retinoid biosynthesis in enterocytes. We provide evidence that the catalytic activity of LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis and maintains optimal retinoid levels in the body.

The Structural and Biochemical Basis of Apocarotenoid Processing by ß-Carotene Oxygenase-2.

In mammals, carotenoids are converted by two carotenoid cleavage oxygenases into apocarotenoids, including vitamin A. Although knowledge about ß-carotene oxygenase-1 (BCO1) and vitamin A metabolism has tremendously increased, the function of ß-carotene oxygenase-2 (BCO2) remains less well-defined. We here studied the role of BCO2 in the metabolism of long chain ß-apocarotenoids, which recently emerged as putative regulatory molecules in mammalian biology. We showed that recombinant murine BCO2 converted the alcohol, aldehyde, and carboxylic acid of a ß-apocarotenoid substrate by oxidative cleavage at position C9,C10 into a ß-ionone and a diapocarotenoid product. Chain length variation (C20 to C40) and ionone ring site modifications of the apocarotenoid substrate did not impede catalytic activity or alter the regioselectivity of the double bond cleavage by BCO2. Isotope labeling experiments revealed that the double bond cleavage of an apocarotenoid followed a dioxygenase reaction mechanism. Structural modeling and site directed mutagenesis identified amino acid residues in the substrate tunnel of BCO2 that are critical for apocarotenoid binding and catalytic processing. Mice deficient for BCO2 accumulated apocarotenoids in their livers, indicating that the enzyme engages in apocarotenoid metabolism. Together, our study provides novel structural and functional insights into BCO2 catalysis and establishes the enzyme as a key component of apocarotenoid homeostasis in mice.

Paracardial fat remodeling affects systemic metabolism through alcohol dehydrogenase 1.

The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.

Carotenoid metabolism at the intestinal barrier.

Carotenoids exert a rich variety of physiological functions in mammals and are beneficial for human health. These lipids are acquired from the diet and metabolized to apocarotenoids, including retinoids (vitamin A and its metabolites). The small intestine is a major site for their absorption and bioconversion. From here, carotenoids and their metabolites are distributed within the body in triacylglycerol-rich lipoproteins to support retinoid signaling in peripheral tissues and photoreceptor function in the eyes. In recent years, much progress has been made in identifying carotenoid metabolizing enzymes, transporters, and binding proteins. A diet-responsive regulatory network controls the activity of these components and adapts carotenoid absorption and bioconversion to the bodily requirements of these lipids. Genetic variability in the genes encoding these components alters carotenoid homeostasis and is associated with pathologies. We here summarize the advanced state of knowledge about intestinal carotenoid metabolism and its impact on carotenoid and retinoid homeostasis of other organ systems, including the eyes, liver, and immune system. The implication of the findings for science-based intake recommendations for these essential dietary lipids is discussed. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

Expression and Characterization of Mammalian Carotenoid Cleavage Dioxygenases.

Carotenoid cleavage dioxygenases (CCDs) are nonheme iron enzymes that catalyze double bond processing of carotenoids and their apocarotenoid metabolites. Mammalian genomes encode three members of this protein family, namely BCO1, BCO2, and RPE65. Mutations and genetic polymorphism in the corresponding genes are associated with inherited blinding diseases, vitamin A deficiency, and high carotenoid plasma levels. Here we describe a method for the heterologous expression of mammalian BCO1 and BCO2 in E. coli and the biochemical characterization of these recombinant enzymes. Dissecting the enzymatic properties of CCDs will advance our knowledge of the biochemical processes that are govern by these disease-associated enzymes and may assist the design of interventions directed against these disease states.

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract.

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in ß- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu2+-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.

Protective role of carotenoids in the visual cycle.

Exposure to light and accumulation of aberrant visual cycle by-products causes stress in the retina. The physical and chemical properties of carotenoids may provide protection against such scenario. These pigments exist in retinas of many vertebrates, including humans. However, the absence of carotenoids in mice, the preferred ophthalmologic animal model, hindered molecular and biochemical examination of the pigments' role in vision. We established a mouse model that accumulates significant amounts of carotenoids in the retina due to inactivating mutations in the Isx and Bco2 genes. We introduced a robust light damage protocol for the mouse retina using green (532 nm) and blue (405 nm) low-energy lasers. We observed that blue but not green laser light treatment triggered the formation of aberrant retinaldehyde isomers in the retina. The production of these visual cycle by-products was accompanied by morphologic damage in inferior parts of the mouse retina. Zeaxanthin supplementation of mice shielded retinoids from these photochemical modifications. These pigments also reduced the extent of the damage to the retina after the blue laser light insult. Thus, our study discovered a novel role of carotenoids in the visual cycle and indicated that vertebrates accumulate carotenoids to shield photoreceptors from short-wavelength light-induced damage.-Widjaja-Adhi, M. A. K., Ramkumar, S., von Lintig, J. Protective role of carotenoids in the visual cycle.

The Biochemical Basis of Vitamin A Production from the Asymmetric Carotenoid ß-Cryptoxanthin.

Vitamin A serves essential functions in mammalian biology as a signaling molecule and chromophore. This lipid can be synthesized from more than 50 putative dietary provitamin A precursor molecules which contain at least one unsubstituted ß-ionone ring. We here scrutinized the enzymatic properties and substrate specificities of the two structurally related carotenoid cleavage dioxygenases (CCDs) which catalyze this synthesis. Recombinant BCO1 split substrates across the C15,C15' double bond adjacent to a canonical ß-ionone ring site to vitamin A aldehyde. Substitution of the ring with a hydroxyl group prevented this conversion. The removal of methyl groups from the polyene carbon backbone of the substrate did not impede enzyme activity. Homology modeling and site-directed mutagenesis identified amino acid residues at the entrance of the substrate tunnel, which determined BCO1's specificity for the canonical ß-ionone ring site. In contrast, BCO2 split substrates across the C9,C10 double bond adjacent to assorted ionone ring sites. Kinetic analysis revealed a higher catalytic efficiency of BCO2 with substrates bearing 3-hydroxy-ß-ionone rings. In the mouse intestine, the asymmetric carotenoid ß-cryptoxanthin with one canonical and one 3-hydroxy-ß-ionone ring site was meticulously converted to vitamin A. The tailoring of this asymmetric substrate occurred by a stepwise processing of the carotenoid substrate by both CCDs and involved a ß-apo-10'-carotenal intermediate. Thus, opposite selectivity for ionone ring sites of the two mammalian CCDs complement each other in the metabolic challenge of vitamin A production from a chemically diverse set of precursor molecules.

Interaction of αA-crystallin F71L mutant with wild type αA- and αB-crystallins by mammalian two hybrid assay.

Incidence of age related cataract (ARC) increases by a variety of factors including metabolic and environmental factors. Nonetheless, genetic mutations are responsible for the altered structural stability of the proteins, especially; the F71L mutation in αA-crystallin has been shown to be responsible for the incidence of cataracts. However, structural characteristics and chaperone function of this mutant and its interaction with wild type (WT) crystallins may aid to decipher its role in cataractogenesis. The aim of the present study is to show the interaction of F71L mutant protein with the WT α-crystallins. The F71L mutant used in this study was created by site-directed mutagenesis, overexpressed and purified. Biophysical characteristics determined by size exclusion HPLC, DLS, CD spectrometry, tryptophan fluorescence and surface hydrophobicity did not show significant structural changes in the mutant protein compared to WT counterpart. Interestingly, the F71L mutant displayed a significant loss in homogenous interaction with WT αA-crystallin and F71L mutant as well as heterogeneous interaction with αB-crystallin as evaluated by mammalian two hybrid system. Our findings suggest that F71L loses the ability to form homo and hetero-oligomers seems to result in the loss in chaperone like activity (CLA) and refractive index resulting in the development of cataracts.

Real-time heterogeneous protein-protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM).

The lens transparency depends on higher concentration of lens proteins and their interactions. α-Crystallin is one of the predominant lens proteins, responsible for proper structural and functional architecture of the lens microenvironment, and any alteration of which results in cataract formation. The R12C, R21L, R49C and R54C are the most significant and prevalent αA-crystallin congenital cataract-causing mutants worldwide. Protein-protein interaction, crucial for lens proper structure and function, was posited to be lost due to point mutation and the elucidation of which could shed light on the molecular basis of cataract. In this conjuncture, we report quartz crystal microbalance (QCM) as a warranted technique for real-time analysis of protein-protein interaction between the N-terminal mutants of αA-crystallin and αB-crystallin. The biophysical characteristics of the mutated proteins were determined by size-exclusion HPLC, far-UV circular dichroism and fluorescence studies. Far-UV circular dichroism spectral analysis displayed slight modifications in ß-sheet of R54C mutant. Altered intrinsic tryptophan fluorescence and decreased bis-ANS fluorescence were observed in all the N-terminal mutations revealing the tertiary structural changes and decreased exposure of surface hydrophobicity. An emphatic fall in the chaperone activity was observed in the N-terminal mutants, R12C, R21L and R54C. QCM analysis revealed the occurrence of strong heterogeneous interaction between αA-crystallin and αB-crystallin. Nevertheless, decreased interactions were observed with the N-terminal mutants. In summary, the present study concludes that the loss of interactions between αA-crystallin N-terminal mutants and αB-crystallin signifies quaternary structural alterations due to mutation in the arginine residues.

Transcriptional regulation of crystallin, redox, and apoptotic genes by C-Phycocyanin in the selenite-induced cataractogenic rat model.

PURPOSE: This study was designed to examine the constrictive potential of C-Phycocyanin (C-PC) in regulating changes imposed on gene expression in the selenite-induced cataract model. METHODS: Wistar rat pups were divided into three groups of eight each. On P10, Group I received an intraperitoneal injection of normal saline. Groups II and III received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight); Group III also received an intraperitoneal injection of C-PC (200 mg/kg bodyweight) on P9-14. Total RNA was isolated on P16, and the relative abundance of mRNA of the crystallin structural genes, redox components, and apoptotic cascade were ascertained with real-time PCR with reference to the internal control ß-actin. RESULTS: Real-time PCR analysis showed the crystallin genes (αA-, ßB1-, γD-) and redox cycle components (Cat, SOD-1, Gpx) were downregulated, the apoptotic components were upregulated, and antiapoptotic Bcl-2 was downregulated in Group II. Treatment with 200 mg/kg bodyweight C-PC (Group III) transcriptionally regulated the instability of the expression of these genes, thus ensuring C-PC is a prospective anticataractogenic agent that probably delays the onset and progression of cataractogenesis induced by sodium selenite. CONCLUSIONS: C-PC treatment possibly prevented cataractogenesis triggered by sodium selenite, by regulating the lens crystallin, redox genes, and apoptotic cascade mRNA expression and thus maintains lens transparency. C-PC may be developed as a potential antioxidant compound applied in the future to prevent and treat age-related cataract.