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1.  Molecular organization and ATP-induced conformational changes of ABCA4, the photoreceptor-specific ABC transporter 
Summary
ATP-binding cassette (ABC) transporters use ATP to translocate various substrates across cellular membranes. Several members of subfamily A of mammalian ABC transporters are associated with severe health disorders, but their unusual complexity and large size have so far precluded structural characterization. ABCA4 is localized to the discs of vertebrate photoreceptor outer segments. This protein transports N-retinylidene-phosphatidylethanolamine to the outer side of disc membranes to prevent formation of toxic compounds causing macular degeneration. An 18 Å-resolution structure of ABCA4 isolated from bovine rod outer segments was determined using electron microscopy and single-particle reconstruction. Significant conformational changes in the cytoplasmic and transmembrane regions were observed upon binding of a non-hydrolysable ATP analogue and accompanied by altered hydrogen/deuterium exchange in the Walker A motif of one of the nucleotide-binding domains. These findings provide an initial view of the molecular organization and functional rearrangements for any member of the ABCA subfamily of ABC transporters.
doi:10.1016/j.str.2013.03.001
PMCID: PMC3654078  PMID: 23562398
2.  THE RHODOPSIN-TRANSDUCIN COMPLEX HOUSES TWO DISTINCT RHODOPSIN MOLECULES 
Journal of structural biology  2013;182(2):164-172.
Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho*, which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1 mM n-dodecyl-β-D-maltoside (DDM) and Rho monomers 5 mM in DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho*•Gt and sConA-Rho*•Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3-D envelope calculated from projections of negatively stained Rho*•Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho*•Gt complexes unequivocally demonstrated a pentameric assembly of the Rho*•Gt complex in which the photoactivated Rho* dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho* dimer in the heteropentameric complex exhibited different capabilities to be regenerated with either 11-cis or 9-cis-retinal.
doi:10.1016/j.jsb.2013.02.014
PMCID: PMC3633645  PMID: 23458690
G protein-coupled receptor; heterotrimeric G protein; photoactivated rhodopsin dimer; transmission electron microscopy; transducin
3.  3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy 
Microscopy  2013;62(1):95-107.
Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.
doi:10.1093/jmicro/dfs091
PMCID: PMC3582345  PMID: 23267047
3D-electron microscopy; detergent belt; membrane protein complex; negative staining; rhodopsin-transducin complex; scanning transmission electron microscopy
4.  Crystal structure of the globular domain of C1QTNF5: Implications for late-onset retinal macular degeneration 
Journal of structural biology  2012;180(3):439-446.
Autosomal dominant late-onset retinal macular degeneration (L-ORMD) is caused by a single S163R mutation in the C1q and tumor necrosis factor-related protein 5 (C1QTNF5) gene. The C1QTNF5 gene encodes a secreted and membrane-associated protein involved in adhesion of retinal pigmented epithelial cells (RPE) to Bruch’s membrane. The crystal structure of the trimeric globular domain of human C1QTNF5 at 1.34 Å resolution reveals unique features of this novel C1q family member. It lacks a Ca2+-binding site, displays a remarkable non-uniform distribution of surface electrostatic potentials and possesses a unique sequence (F181F182G183G184W185P186) that forms a hydrophobic plateau surrounded by Lys and Arg residues with a solvent cavity underneath. S163 forms a hydrogen bond with F182 in a hydrophobic area extending to the hydrophobic plateau. The pathogenic mutation S163R disrupts this hydrogen bonding and positively charges these hydrophobic areas. Thus, our analysis provides insights into the structural basis of the L-ORMD disease mechanism.
doi:10.1016/j.jsb.2012.07.011
PMCID: PMC3496058  PMID: 22892318
L-ORMD; L-ORD; age-related macular degeneration; AMD; CTRP5; drusen
5.  Systems pharmacology identifies drug targets for Stargardt disease–associated retinal degeneration 
The Journal of Clinical Investigation  2013;123(12):5119-5134.
A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.
doi:10.1172/JCI69076
PMCID: PMC3859412  PMID: 24231350
6.  Retinyl Ester Formation by Lecithin:Retinol Acyltransferase Is a Key Regulator of Retinoid Homeostasis in Mouse Embryogenesis* s 
The Journal of biological chemistry  2007;283(9):5611-5621.
The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids (vitamin A and its metabolites). The mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of suboptimal or excessive maternal dietary vitamin A intake have not been established. We investigated the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Dams lacking both LRAT and retinol-binding protein (RBP), the sole specific carrier for retinol in serum, were maintained on diets containing different amounts of vitamin A during pregnancy. We hypothesized that the lack of both proteins would make the embryo more vulnerable to changes in maternal dietary vitamin A intake. Our data demonstrate that maternal dietary vitamin A deprivation during pregnancy generates a severe retinoid-deficient phenotype of the embryo due to the severe retinoid-deficient status of the double mutant dams rather than to the lack of LRAT in the developing tissues. Moreover, in the case of excessive maternal dietary vitamin A intake, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with STRA6, the recently identified cell surface receptor for retinol-RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues. In contrast, the pathway of retinoic acid synthesis does not contribute significantly to regulating retinoid homeostasis during mammalian development except under conditions of severe maternal retinoid deficiency.
doi:10.1074/jbc.M708885200
PMCID: PMC3771510  PMID: 18093970
7.  In vivo two-photon imaging of the mouse retina 
Biomedical Optics Express  2013;4(8):1285-1293.
Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.
doi:10.1364/BOE.4.001285
PMCID: PMC3756587  PMID: 24009992
(330.4460) Ophthalmic optics and devices; (180.4315) Nonlinear microscopy; (170.0110) Imaging systems
8.  Substrate–Induced Changes in Dynamics of Rhodopsin Kinase (G Protein–Coupled Receptor Kinase 1)† 
Biochemistry  2012;51(16):3404-3411.
G protein–coupled receptor (GPCR) kinases (GRKs) instigate the desensitization of activated GPCRs via phosphorylation that promotes interaction with arrestins, thereby preventing the interaction of GPCRs with heterotrimeric G proteins. A current proposed model of GRK1 activation involves the binding of activated rhodopsin (Rho*) to the N–terminal region of GRK1. Perhaps concomitantly, this N–terminal region also stabilizes a closed, active conformation of the kinase domain. To further probe this model, we mapped changes in the backbone flexibility of GRK1 as it binds to its two substrates, adenosine triphosphate (Mg2+·ATP) and Rho*. We found that the conformational flexibility of GRK1 was reduced in the presence of either Mg2+·ATP and/or Rho*, with Mg2+·ATP having the greatest effect. In a truncated form of GRK1 lacking the N–terminal region (ΔN–GRK1), peptides that directly interact with ATP were not as dramatically stabilized by adding Mg2+·ATP, and dynamics were greater in the interface between the large lobe of the kinase domain and the regulator of G protein signaling homology domain. In the presence of Mg2+·ATP, the influence of Rho* versus Rho was negligible on GRK1 dynamics.
doi:10.1021/bi300295y
PMCID: PMC3422436  PMID: 22480180
9.  Retinal cone and rod photoreceptor cells exhibit differential susceptibility to light–induced damage 
Journal of Neurochemistry  2012;121(1):146-156.
All-trans-retinal and its condensation-products can cause retinal degeneration in a light–dependent manner and contribute to the pathogenesis of human macular diseases such as Stargardt’s disease and age–related macular degeneration (AMD). Although these toxic retinoid by–products originate from rod and cone photoreceptor cells, the contribution of each cell type to light–induced retinal degeneration is unknown. Here the primary objective was to learn whether rods or cones are more susceptible to light–induced, all–trans–retinal–mediated damage. Previously, we reported that mice lacking enzymes that clear all–trans–retinal from the retina, ATP–binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), manifested light-induced retinal dystrophy. We first examined early-stage-AMD patients and found retinal degenerative changes in rod-rich rather than cone-rich regions of the macula. We then evaluated transgenic mice with rod–only and cone–like–only retinas in addition to progenies of such mice inbred with Rdh8−/− Abca4−/− mice. Of all these strains, Rdh8−/− Abca4−/− mice with a mixed rod–cone population showed the most severe retinal degeneration under regular cyclic light conditions. Intense light exposure induced acute retinal damage in Rdh8−/− Abca4−/− and rod–only mice but not cone–like–only mice. These findings suggest that progression of retinal degeneration in Rdh8-/- Abca4-/- mice is affected by differential vulnerability of rods and cones to light.
doi:10.1111/j.1471-4159.2012.07647.x
PMCID: PMC3303932  PMID: 22220722
visual cycle; photoreceptor; retinoid; retina; Stargardt’s disease; age-related macular degeneration
10.  Light-Induced Translocation of RGS9-1 and Gβ5L in Mouse Rod Photoreceptors 
PLoS ONE  2013;8(3):e58832.
The transducin GTPase-accelerating protein complex, which determines the photoresponse duration of photoreceptors, is composed of RGS9-1, Gβ5L and R9AP. Here we report that RGS9-1 and Gβ5L change their distribution in rods during light/dark adaptation. Upon prolonged dark adaptation, RGS9-1 and Gβ5L are primarily located in rod inner segments. But very dim-light exposure quickly translocates them to the outer segments. In contrast, their anchor protein R9AP remains in the outer segment at all times. In the dark, Gβ5L's interaction with R9AP decreases significantly and RGS9-1 is phosphorylated at S475 to a significant degree. Dim light exposure leads to quick de-phosphorylation of RGS9-1. Furthermore, after prolonged dark adaptation, RGS9-1 and transducin Gα are located in different cellular compartments. These results suggest a previously unappreciated mechanism by which prolonged dark adaptation leads to increased light sensitivity in rods by dissociating RGS9-1 from R9AP and redistributing it to rod inner segments.
doi:10.1371/journal.pone.0058832
PMCID: PMC3610756  PMID: 23555598
11.  A Microparticle/Hydrogel Combination Drug-Delivery System for Sustained Release of Retinoids 
Purpose.
To design and develop a drug-delivery system containing a combination of poly(d,l-lactide-co-glycolide) (PLGA) microparticles and alginate hydrogel for sustained release of retinoids to treat retinal blinding diseases that result from an inadequate supply of retinol and generation of 11-cis-retinal.
Methods.
To study drug release in vivo, either the drug-loaded microparticle–hydrogel combination was injected subcutaneously or drug-loaded microparticles were injected intravitreally into Lrat−/− mice. Orally administered 9-cis-retinoids were used for comparison and drug concentrations in plasma were determined by HPLC. Electroretinography (ERG) and both chemical and histologic analyses were used to evaluate drug effects on visual function and morphology.
Results.
Lrat−/− mice demonstrated sustained drug release from the microparticle/hydrogel combination that lasted 4 weeks after subcutaneous injection. Drug concentrations in plasma of the control group treated with the same oral dose rose to higher levels for 6−7 hours but then dropped markedly by 24 hours. Significantly increased ERG responses and a markedly improved retinal pigmented epithelium (RPE)–rod outer segment (ROS) interface were observed after subcutaneous injection of the drug-loaded delivery combination. Intravitreal injection of just 2% of the systemic dose of drug-loaded microparticles provided comparable therapeutic efficacy.
Conclusions.
Sustained release of therapeutic levels of 9-cis-retinoids was achieved in Lrat−/− mice by subcutaneous injection in a microparticle/hydrogel drug-delivery system. Both subcutaneous and intravitreal injections of drug-loaded microparticles into Lrat−/− mice improved visual function and retinal structure.
A novel drug-delivery system was developed for sustained release of therapeutic levels of 9-cis-retinoids. A PLGA microsphere alginate hydrogel combination was used both in vitro and in vivo to evaluate its therapeutic efficacy in retinas of Lrat−/− mice.
doi:10.1167/iovs.12-10279
PMCID: PMC3465014  PMID: 22918645
12.  Imaging of protein crystals with two–photon microscopy† 
Biochemistry  2012;51(8):1625-1637.
Second–order non–linear optical imaging of chiral crystals (SONICC), that portrays second harmonic generation (SHG) by non–centrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal–to–noise ratio. Here we report the incorporation of both SONICC and two–photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ~10 μm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG–silent protein crystals. Our experimental data suggests that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPFE. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with non–damaging infrared light in a single system, makes the combination of SHG and intrinsic visible TPEF a powerful tool for non–destructive protein crystal identification and characterization during crystallization trials.
doi:10.1021/bi201682q
PMCID: PMC3293215  PMID: 22324807
13.  Tetramerization of SAMHD1 Is Required for Biological Activity and Inhibition of HIV Infection* 
The Journal of Biological Chemistry  2013;288(15):10406-10417.
Background: SAMHD1, a dGTP-activated dNTPase, inhibits retrovirus infection at the reverse transcription step in monocytes and quiescent T lymphocytes.
Results: dGTP-induced SAMHD1 tetramerization correlates with its functional activation.
Conclusion: SAMHD1 tetramers are the biologically active form of this dNTPase.
Significance: Learning how SAMHD1 function is regulated is important for understanding innate and anti-viral immunity.
SAMHD1 is a dGTP-activated dNTPase that has been implicated as a modulator of the innate immune response. In monocytes and their differentiated derivatives, as well as in quiescent cells, SAMHD1 strongly inhibits HIV-1 infection and, to a lesser extent, HIV-2 and simian immunodeficiency virus (SIV) because of their virion-associated virulence factor Vpx, which directs SAMHD1 for proteasomal degradation. Here, we used a combination of biochemical and virologic approaches to gain insights into the functional organization of human SAMHD1. We found that the catalytically active recombinant dNTPase is a dGTP-induced tetramer. Chemical cross-linking studies revealed SAMHD1 tetramers in human monocytic cells, in which it strongly restricts HIV-1 infection. The propensity of SAMHD1 to maintain the tetrameric state in vitro is regulated by its C terminus, located outside of the catalytic domain. Accordingly, we show that the C terminus is required for the full ability of SAMHD1 to deplete dNTP pools and to inhibit HIV-1 infection in U937 monocytes. Interestingly, the human SAMHD1 C terminus contains a docking site for HIV-2/SIVmac Vpx and is known to have evolved under positive selection. This evidence indicates that Vpx targets a functionally important element in SAMHD1. Together, our findings imply that SAMHD1 tetramers are the biologically active form of this dNTPase and provide new insights into the functional organization of SAMHD1.
doi:10.1074/jbc.M112.443796
PMCID: PMC3624423  PMID: 23426366
HIV; Innate Immunity; Protein Chemistry; Protein Complexes; Virus
14.  Post–translational Modifications of Serotonin Type 4 Receptor Heterologously Expressed in Mouse Rod Cells† 
Biochemistry  2011;51(1):214-224.
The G–protein–coupled serotonin receptor type 4 (5–HT4R) is a pharmacological target implicated in a variety of gastro-intestinal and nervous system disorders. Like many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post–translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin’s biosynthetic machinery in rod cells of the retina to express heterologous G–protein coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5–HT4R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5–HT4R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic (TG) proteins, but also the complications that non–native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.
doi:10.1021/bi201707v
PMCID: PMC3268512  PMID: 22145929
15.  Key enzymes of the retinoid (visual) cycle in vertebrate retina 
Biochimica et biophysica acta  2011;1821(1):137-151.
A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases.
doi:10.1016/j.bbalip.2011.03.005
PMCID: PMC3158816  PMID: 21447403
RPE65; retinol dehydrogenase; RDH; visual cycle; retinoid cycle; isomerization; retinol; retinal; retina; retinoid isomerase; retinal degeneration; photoreceptors; lecithin:retinol acyltransferase; chromophore; LRAT
16.  Primary amines protect against retinal degeneration in mouse models of retinopathies 
Nature chemical biology  2011;8(2):170-178.
Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore, 11-cis-retinal, and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomered product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing FDA-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by mass spectrometry. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that displays features of Stargardt’s and age-related retinal degeneration.
doi:10.1038/nchembio.759
PMCID: PMC3518042  PMID: 22198730
Photoreceptor cells; A2E; RPE; retina; Stargardt’s disease; age-related macular degeneration; retinal degeneration; retinal condensation products
17.  Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization 
The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin’s stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration.
doi:10.1172/JCI66176
PMCID: PMC3533307  PMID: 23221340
18.  RHODOPSIN-TRANSDUCIN HETEROPENTAMER: THREE-DIMENSIONAL STRUCTURE AND BIOCHEMICAL CHARACTERIZATION 
Journal of structural biology  2011;176(3):387-394.
The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho* form. Rho* binds the heterotrimeric G protein, transducin (Gt) inducing GDP to GTP exchange and Gt dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho*•Gt complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho* to Gt in the complex and its mass determined by scanning transmission electron microscopy was 221±12 kDa. A 21.6Å structure then was calculated from projections of negatively stained Rho*•Gt complexes. The molecular envelope thus determined accommodated two Rho molecules together with one Gt heterotrimer, corroborating the heteropentameric structure of the Rho*•Gt complex.
doi:10.1016/j.jsb.2011.08.016
PMCID: PMC3210324  PMID: 21925606
G protein-coupled receptor; heterotrimeric G protein; photoactivated rhodopsin; scanning transmission electron microscopy; transducin
19.  Detergents Stabilize the Conformation of Phosphodiesterase 6† 
Biochemistry  2011;50(44):9520-9531.
Membrane–bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and β subunit catalytic core, interactions of the core with two intrinsically–disordered, proteolysis–prone inhibitory PDEγ (Pγ) subunits, and binding of two isoprenyl–binding proteins δ, called PrBP/δ, to the isoprenylated C–termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by lack of a heterologous expression system for the holo–enzyme. In this work, we purified PDE6 in the presence of PrBP/δ and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin–activated enzyme. Some detergents removed PrBP/δ from the PDE complex, separating it from the holo–enzyme after PDE6 purification. Additionally, selected detergents also significantly reduced dissociation of PDE6 subunits, increasing its homogeneity, and stabilizing the holo–enzyme by substituting for its native membrane environment.
doi:10.1021/bi2014695
PMCID: PMC3215081  PMID: 21978030
20.  The physiological impact of microRNA gene regulation in the retina 
microRNAs (miRNAs) are small, stable RNA molecules that post-transcriptionally regulate gene expression in plants and animals by base pairing to partially complementary sequences on target mRNAs to inhibit protein synthesis. More than 250 miRNAs are reportedly expressed in the retina, and miRNA gene regulation has been shown to affect retinal development, function, and disease. Here we highlight recent advances in understanding the functional roles of vertebrate retinal miRNAs. Details are emerging about the physiological impact of specific miRNAs in the developing and mature retina, and we discuss a group of emerging technologies for studying miRNAs, which can be employed to yield a deeper understanding of retinal miRNA gene regulation.
doi:10.1007/s00018-012-0976-7
PMCID: PMC3443797  PMID: 22460583
microRNA; Retina; Dicer; Photoreceptor; Argonaute
21.  Inflammatory priming predisposes mice to age-related retinal degeneration 
The Journal of Clinical Investigation  2012;122(8):2989-3001.
Disruption of cellular processes affected by multiple genes and accumulation of numerous insults throughout life dictate the progression of age-related disorders, but their complex etiology is poorly understood. Postmitotic neurons, such as photoreceptor cells in the retina and epithelial cells in the adjacent retinal pigmented epithelium, are especially susceptible to cellular senescence, which contributes to age-related retinal degeneration (ARD). The multigenic and complex etiology of ARD in humans is reflected by the relative paucity of effective compounds for its early prevention and treatment. To understand the genetic differences that drive ARD pathogenesis, we studied A/J mice, which develop ARD more pronounced than that in other inbred mouse models. Although our investigation of consomic strains failed to identify a chromosome associated with the observed retinal deterioration, pathway analysis of RNA-Seq data from young mice prior to retinal pathological changes revealed that increased vulnerability to ARD in A/J mice was due to initially high levels of inflammatory factors and low levels of homeostatic neuroprotective factors. The genetic signatures of an uncompensated preinflammatory state and ARD progression identified here aid in understanding the susceptible genetic loci that underlie pathogenic mechanisms of age-associated disorders, including several human blinding diseases.
doi:10.1172/JCI64427
PMCID: PMC3408755  PMID: 22797304
22.  Role of Membrane Integrity on G protein-coupled Receptors: Rhodopsin Stability and Function 
Progress in lipid research  2011;50(3):267-277.
Rhodopsin is a prototypical G protein-coupled receptor (GPCR) – a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes may result in visual dysfunction and blindness.
doi:10.1016/j.plipres.2011.03.002
PMCID: PMC3114187  PMID: 21435354
G protein-coupled receptor(s); rhodopsin; transducin (Gt); photoreceptor; rod outer segment; membranes; oligomerization; signal transduction; phospholipids; cholesterol; phospholipase A2
23.  Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors 
Biochemistry  2011;50(25):5590-5600.
Mouse photoreceptor function and survival critically depend on Ca2+-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12 and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5 to 28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 μM cGMP s−1 or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca2+ in the presence of GCAP1 (EC50Ca ~132-139 nM) than GCAP2 (EC50Ca ~50-59 nM), thus arguing that Ca2+ sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.
doi:10.1021/bi200491b
PMCID: PMC3127287  PMID: 21598940
photoreceptors; guanylyl cyclase; guanylyl cyclase activating proteins; cGMP; retina
24.  Serial sectioning for examination of photoreceptor cell architecture by focused ion beam technology 
Journal of neuroscience methods  2011;198(1):70-76.
Structurally deciphering complex neural networks requires technology with sufficient resolution to allow visualization of single cells and their intimate surrounding connections. Scanning electron microscopy (SEM), coupled with serial ion ablation (SIA) technology, presents a new avenue to study these networks. SIA allows ion ablation to remove nanometer sections of tissue for SEM imaging, resulting in serial section data collection for three-dimensional reconstruction. Here we highlight a method for preparing retinal tissues for imaging of photoreceptors by SIA-SEM technology. We show that this technique can be used to visualize whole rod photoreceptors and the internal disc elements from wild-type (wt) mice. The distance parameters of the discs and photoreceptors are in good agreement with previous work with other methods. Moreover, we show that large planes of retinal tissue can be imaged at high resolution to display the packing of normal rods. Finally, SIA-SEM imaging of retinal tissue from a mouse model (Nrl−/−) with phenotypic changes akin to the human disease enhanced S-cone syndrome (ESCS) revealed a structural profile of overall photoreceptor ultrastructure and internal elements that accompany this disease. Overall, this work presents a new method to study photoreceptor cells at high structural resolution that has a broad applicability to the visual neuroscience field.
doi:10.1016/j.jneumeth.2011.03.013
PMCID: PMC3090522  PMID: 21439323
Cone photoreceptor; enhanced S-cone syndrome; focused ion beam; Nrl; retina; rod photoreceptor; serial ion ablation; vision
25.  Heterogeneous N-terminal Acylation of Retinal Proteins Results from the Retina’s Unusual Lipid Metabolism†,§ 
Biochemistry  2011;50(18):3764-3776.
Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1 and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were: C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca2+-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates; C14:2- and C14:1-coenyzme A to C14:0-coenzyme A were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A.
doi:10.1021/bi200245t
PMCID: PMC3086940  PMID: 21449552

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