Marcin Golczak

Crystal structure of a photoactivated deprotonated intermediate of rhodopsin

The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors.

Retinopathy in Mice Induced by Disrupted All-trans-retinal Clearance

The visual (retinoid) cycle is a fundamental metabolic process in vertebrate retina responsible for production of 11-cis-retinal, the chromophore of rhodopsin and cone pigments. 11-cis-Retinal is bound to opsins, forming visual pigments, and when the resulting visual chromophore 11-cis-retinylidene is photoisomerized to all-trans-retinylidene, all-trans-retinal is released from these receptors. Toxic byproducts of the visual cycle formed from all-trans-retinal often are associated with lipofuscin deposits in the retinal pigmented epithelium (RPE), but it is not clear whether aberrant reactions of the visual cycle participate in RPE atrophy, leading to a rapid onset of retinopathy. Here we report that mice lacking both the ATP-binding cassette transporter 4 (Abca4) and enzyme retinol dehydrogenase 8 (Rdh8), proteins critical for all-trans-retinal clearance from photoreceptors, developed severe RPE/photoreceptor dystrophy at an early age. This phenotype includes lipofuscin, drusen, and basal laminar deposits, Bruchs membrane thickening, and choroidal neovascularization. Importantly, the severity of visual dysfunction and retinopathy was exacerbated by light but attenuated by treatment with retinylamine, a visual cycle inhibitor that slows the flow of all-trans-retinal through the visual cycle. These findings provide direct evidence that aberrant production of toxic condensation byproducts of the visual cycle in mice can lead to rapid, progressive retinal degeneration.

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.

A mitochondrial enzyme degrades carotenoids and protects against oxidative stress

Carotenoids are the precursors for vitamin A and are proposed to prevent oxidative damage to cells. Mammalian genomes encode a family of structurally related nonheme iron oxygenases that modify double bonds of these compounds by oxidative cleavage and cis‐to‐trans isomerization. The roles of the family members BCMO1 and RPE65 for vitamin A production and vision have been well established. Surprisingly, we found that the third family member, β,β‐carotene‐9’,10’‐oxygenase (BCDO2), is a mitochondrial carotenoid‐oxygenase with broad substrate specificity. In BCDO2‐deficient mice, carotenoid homeostasis was abrogated, and carotenoids accumulated in several tissues. In hepatic mitochondria, accumulated carotenoids induced key markers of mitochondrial dysfunction, such as manganese superoxide dismutase (9‐fold), and reduced rates of ADP‐dependent respiration by 30%. This impairment was associated with an 8‐ to 9‐fold induction of phosphor‐MAP kinase and phosphor‐AKT, markers of cell signaling pathways related to oxidative stress and disease. Administration of carotenoids to human HepG2 cells depolarized mitochondrial membranes and resulted in the production of reactive oxygen species. Thus, our studies in BCDO2‐deficient mice and human cell cultures indicate that carotenoids can impair respiration and induce oxidative stress. Mammalian cells thus express a mitochondrial carotenoid‐oxygenase that degrades carotenoids to protect these vital organelles.—Amengual, J., Lobo, G. P., Golczak, M., Li, H. N. M., Klimova, T., Hoppel, C. L., Wyss, A., Palczewski, K., von Lintig, J. A mitochondrial enzyme degrades carotenoids and protects against oxidative stress. FASEB J. 25, 948–959 (2011). www.fasebj.org

RBP4 Disrupts Vitamin A Uptake Homeostasis in a STRA6-Deficient Animal Model for Matthew-Wood Syndrome

The cellular uptake of vitamin A from its RBP4-bound circulating form (holo-RBP4) is a homeostatic process that evidently depends on the multidomain membrane protein STRA6. In humans, mutations in STRA6 are associated with Matthew-Wood syndrome, manifested by multisystem developmental malformations. Here we addressed the metabolic basis of this inherited disease. STRA6-dependent transfer of retinol from RBP4 into cultured NIH 3T3 fibroblasts was enhanced by lecithin:retinol acyltransferase (LRAT). The retinol transfer was bidirectional, strongly suggesting that STRA6 acts as a retinol channel/transporter. Loss-of-function analysis in zebrafish embryos revealed that Stra6 deficiency caused vitamin A deprivation of the developing eyes. We provide evidence that, in the absence of Stra6, holo-Rbp4 provokes nonspecific vitamin A excess in several embryonic tissues, impairing retinoic acid receptor signaling and gene regulation. These fatal consequences of Stra6 deficiency, including craniofacial and cardiac defects and microphthalmia, were largely alleviated by reducing embryonic Rbp4 levels by morpholino oligonucleotide or pharmacological treatments.

Human cone photoreceptor dependence on RPE65 isomerase

The visual (retinoid) cycle, the enzymatic pathway that regenerates chromophore after light absorption, is located primarily in the retinal pigment epithelium (RPE) and is essential for rod photoreceptor survival. Whether this pathway also is essential for cone photoreceptor survival is unknown, and there are no data from man or monkey to address this question. The visual cycle is naturally disrupted in humans with Leber congenital amaurosis (LCA), which is caused by mutations in RPE65, the gene that encodes the retinoid isomerase. We investigated such patients over a wide age range (3–52 years) for effects on the cone-rich human fovea. In vivo microscopy of the fovea showed that, even at the youngest ages, patients with RPE65-LCA exhibited cone photoreceptor loss. This loss was incomplete, however, and residual cone photoreceptor structure and function persisted for decades. Basic questions about localization of RPE65 and isomerase activity in the primate eye were addressed by examining normal macaque. RPE65 was definitively localized by immunocytochemistry to the central RPE and, by immunoblotting, appeared to concentrate in the central retina. The central retinal RPE layer also showed a 4-fold higher retinoid isomerase activity than more peripheral RPE. Early cone photoreceptor losses in RPE65-LCA suggest that robust RPE65-based visual chromophore production is important for cones; the residual retained cone structure and function support the speculation that alternative pathways are critical for cone photoreceptor survival.

Chemistry of the Retinoid (Visual) Cycle

As succinctly summarized by Wolf,1 lack of vitamin A (all-trans-retinol) was recognized by ancient Egyptians as causing a visual deficiency involving the retina and cornea that could be cured by eating liver. One of the symptoms of vitamin A deficiency is night blindness or nyctalopia (from Greek νύκτ-, nykt – night; and αλαός, alaos – blindness), recognized by ancient Greeks, including Hippocrates, as affecting the retina.2 In 1913 McCollum showed that “fat-soluble factor A” was essential for growth of a rat colony (reviewed in ref (3)). The treatment of factor A-deficiency included liver or liver extracts, but later in 1930 Moore found that yellow pigment (carotene) was a good substitute for this therapy.4 A major breakthrough occurred in 1931 when the chemical structures for β,β-carotene and retinol (its all-trans isomer now known as vitamin A) were determined by Karrer and colleagues.5 But, it was Wald who discovered that retinol derivatives constitute the chemical basis of our vision,6 a contribution subsequently recognized by a Nobel Prize award in 1967. In 1950–1960, a variety of vitamin A metabolic transformations, including oxidation/reduction and esterification, were elucidated by Olson,7−20 Goodman,21−32 Chytil and Ong,33−41 and Norum and Blomhoff.42−57 The discovery that one set of these metabolites, namely retinoic acids, plays a key role in the nuclear regulation of a large number of genes added a notable dimension to our knowledge of gene expression. This mechanism is also a critical player in the successful healing of corneal wounds,58 a second manifestation of vitamin A-deficiency recognized earlier. Further progress in understanding the multiple physiological roles of retinoids has been made in recent years, due mainly to the successful application of modern scientific technology. Examples include enzymology combined with structural biology, in vivo imaging based on retinoid fluorescence, improvements in analytical methods, generation and testing of animal models of human diseases with specific pathogenic genetics, genetic analysis of human conditions related to changes in vitamin A metabolism, and pharmacological approaches to combat these diseases. In this review we focus on the involvement of retinoids in supporting vision via light-sensitive rod and cone photoreceptor cells in the retina. We begin with a brief description of isopentenyl diphosphate (IPP) biosynthesis, which is essential for carotenoid (C40 isoprenoid) production. Certain of these colored compounds, such as lutein, are deposited in our retina’s macula, appearing as a “yellow” spot. Other carotenoids containing at least one unmodified β-ionone ring (represented by β,β-carotene and cryptoxanthin) serve as precursors of all-trans-retinal. Many different compounds can be generated from this monocyclic diterpenoid, which contains a β-ionone ring and polyene chain with a C15 aldehyde group. Among the numerous enzymatic activities that contribute to retinoid metabolism, polyene trans/cis isomerization is a particularly fascinating reaction that occurs in specialized structures of the retina based on a two cell system comprised of retinal photoreceptor cells and the retinal pigment epithelium (RPE). A specific enzyme system, called the retinoid (visual) cycle, has evolved to accomplish retinoid isomerization that is required for visual function in vertebrates. Individual enzymes of this pathway harbor secrets about the molecular mechanisms of this chemical transformation. Malfunctions of these processes or other pathological reactions often precipitate severe retinal pathologies. This review attempts to balance contributions that have been published over the past decades and does not intend to replace the views of investigators with different perspectives of retinoid chemistry in the eye.59−85

Probing Mechanisms of Photoreceptor Degeneration in a New Mouse Model of the Common Form of Autosomal Dominant Retinitis Pigmentosa due to P23H Opsin Mutations

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1–10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development.

Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-Å resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.

Mechanism of All-trans-retinal Toxicity with Implications for Stargardt Disease and Age-related Macular Degeneration

Background: High levels of all-trans-retinal (atRAL) are associated with photoreceptor degeneration. Results: atRAL promotes NADPH oxidase-mediated overproduction of intracellular reactive oxygen species. Conclusion: A cascade of signaling events is demonstrated to underlie the action of atRAL in photoreceptor degeneration in mice. Significance: Mechanistic elucidation of atRAL-mediated photoreceptor degeneration is essential for understanding the molecular pathogenesis of Stargardt disease and other types of retinal degeneration. Compromised clearance of all-trans-retinal (atRAL), a component of the retinoid cycle, increases the susceptibility of mouse retina to acute light-induced photoreceptor degeneration. Abca4−/−Rdh8−/− mice featuring defective atRAL clearance were used to examine the one or more underlying molecular mechanisms, because exposure to intense light causes severe photoreceptor degeneration in these animals. Here we report that bright light exposure of Abca4−/−Rdh8−/− mice increased atRAL levels in the retina that induced rapid NADPH oxidase-mediated overproduction of intracellular reactive oxygen species (ROS). Moreover, such ROS generation was inhibited by blocking phospholipase C and inositol 1,4,5-trisphosphate-induced Ca2+ release, indicating that activation occurs upstream of NADPH oxidase-mediated ROS generation. Because multiple upstream G protein-coupled receptors can activate phospholipase C, we then tested the effects of antagonists of serotonin 2A (5-HT2AR) and M3-muscarinic (M3R) receptors and found they both protected Abca4−/−Rdh8−/− mouse retinas from light-induced degeneration. Thus, a cascade of signaling events appears to mediate the toxicity of atRAL in light-induced photoreceptor degeneration of Abca4−/−Rdh8−/− mice. A similar mechanism may be operative in human Stargardt disease and age-related macular degeneration.