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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProtein localization was assayed by immunohistochemistry fromretinal sections and by production of transgenic Xenopus laevistadpoles expressing EGFP-rab11a and/or mCherry-NUDC.Results: NUDC was identified as a protein that bound rhodopsin inassociation with rab11a. Reciprocal pull-down experimentsconfirmed the association between rhodopsin, rab11a, and NUDC.Rhodopsin C-terminal mutants Q344Ter and rhodopsin-EGFP boundneither rab11a nor NUDC. Immunohistochemistry revealed NUDClocalization in the inner segment of photoreceptors. Examination oftransgenic mCherry-NUDC expressing tadpoles revealed a punctatedistribution in the inner segment with minimal outer segmentlocalization. In tadpoles expressing EGFP-rab11a and mCherry-NUDC, there is overlap between EGFP and mCherry positive punctain the inner segment, in a distribution between the Golgi apparatusand the base of the outer segment.Conclusions: NUDC was shown to interact with rab11a andrhodopsin in preparations from mouse retina. This interaction wasabolished in two mouse lines expressing either truncated or extendedrhodopsin C-termini. Immunohistochemical and transgenicexpression experiments reveal a role for NUDC in rab11a positivevesicular trafficking in the inner segment.Commercial Relationships: Alecia K. Gross, None; Nicholas J.Reish, NoneSupport: NIH Grant EY019311, The EyeSight Foundation ofAlabama, The Karl Kirchgessner Foundation, E. Matilda ZieglerFoundationProgram Number: 1225Presentation Time: 9:00 AM - 9:15 AMAddressing the Role of PhLP in the Folding and Assembly of G-protein βγ dimerMaxim Sokolov, Satyabrata Sinha, Marycharmain Belcastro, XueliGao. Ophthalmology, West Virginia Univ Eye Institute,Morgantown, WV.Purpose: To get better understanding of the roles of the cytosolicchaperonin containing T-complex protein 1 (CCT) and its co-factorphosducin-like protein (PhLP) in the folding and assembly of theheterotrimeric G-protein βγ subunits complex.Methods: The interaction of PhLP and its short splice isoform(PhLPs) with CCT and G protein subunits was studied in both mousephotoreceptors and cell culture. The levels of mRNA and proteinswere determined by quantitative real-time PCR and Western blotting,respectively. The assembly of epitope-tagged Gβ1 and Gγ1 subunitswas monitored using a pull down assay.Results: We found that both PhLP and PhLPs form a complex withCCT, however, only PhLP facilitated the assembly of the Gβ1γ1dimer. Interestingly, while doing so, PhLP interacted primarily withGβ1 and not with the Gβ1γ1 complex. In contrast, PhLPs, whichlacks an important Gβ-binding domain, showed a tendency to form astable and apparently inactive tertiary complex with both CCT andthe Gβ1 trapped inside. Such a mode of action was consistent withthe pronounced cytotoxicity of PhLPs in rod photoreceptorsexpressing high levels of Gβ1. As a result, the levels of Gβ1 and,contingent on it, Gγ1 in rods became significantly reduced. Rathersurprisingly, Gαt1 became down-regulated under this circumstanceon the transcriptional level.Conclusions: Our data suggest that PhLP assists G proteinbiosynthesis by shuttling newly folded Gβ1 away from thechaperonin, priming it for binding with Gγ1, which in turn, triggersPhLP’s own release. A splice isoform of PhLP, however, hinders thisprocess via a complex mechanism.Commercial Relationships: Maxim Sokolov, None; SatyabrataSinha, None; Marycharmain Belcastro, None; Xueli Gao, NoneSupport: NIH Grant EY019665, RPBProgram Number: 1226Presentation Time: 9:15 AM - 9:30 AMLight-Dependent Phosphorylation of BBS5 in Photoreceptorsand Its Interaction with Arrestin1Tyler S. Smith, Donald R. Dugger, Susan N. Bolch, J HughMcDowell, W Clay Smith. Ophthalmology, University of Florida,Gainesville, FL.Purpose: We have previously demonstrated that Bardet-BiedlSyndrome 5 (BBS5) protein is one member of the phosphoproteomein photoreceptors. Here we provide an analysis of the kinase involvedin BBS5 phosphorylation and investigate the functional effect ofBBS5 phosphorylation on its interaction with other proteins.Methods: BBS5 was cloned from murine retinal mRNA, andheterologously expressed in bacteria with either a His(6) tag or as afusion protein with glutathione-S-transferase (GST). BBS5 wasphosphorylated in situ by endogenous kinases; phosphorylation ofBBS5 was tested in vitro using a panel of six kinases. Interaction ofBBS5 with arrestin was assessed in vitro via immunoprecipitation.Tissue localization of BBS5 was examined in cryosections ofXenopus laevis retina using a monoclonal antibody prepared againstheterologously expressed BBS5.Results: BBS5 is phosphorylated in a light-dependent manner,showing a threshold of phosphorylation that correlates with theinitiation of arrestin1 translocation. Phosphorylation of BBS5 canalso be stimulated in vivo by phorbol-12,13-diacetate, a proteinkinase C agonist. In vitro analysis demonstrates that BBS5 isphosphorylated by PKC, but not by protein kinase A, cGMPdependentprotein kinase, casein kinase I or II, orcalcium/calmodulin-dependent protein kinase II. Arrestin1 coimmunoprecipitateswith BBS5/GST; phosphorylation of BBS5reduces the interaction between arrestin1 and BBS5. Under identicalconditions, arrestin1 shows negligible co-immunoprecipitation withBBS8. In photoreceptors, BBS5 principally localizes to the axonemesof rods and cones, photoreceptor inner segments, and synapticregions.Conclusions: Phosphorylation of BBS5 in retina is initiated by light,apparently through PKC activation based on in vitro studies.Activation of PKC in situ using phorbol ester leads to BBS5phosphorylation, supporting this conclusion. The three-foldobservation that (1) BBS5 directly interacts with arrestin1, (2) thatphosphorylation of BBS5 reduces the affinity of arrestin1 for BBS5,and (3) that BBS5 principally localizes to the axoneme suggests thatphosphorylation of BBS5 may play a role in regulating lightdependenttranslocation of arrestin1.Commercial Relationships: Tyler S. Smith, None; Donald R.Dugger, None; Susan N. Bolch, None; J Hugh McDowell, None; WClay Smith, NoneSupport: NIH Grants EY014864, EY006225, EY021721 andResearch to Prevent BlindnessProgram Number: 1227Presentation Time: 9:30 AM - 9:45 AMThe Essential Role of CCT in Outer Segment MorphogenesisSatyabrata Sinha 1 , Marycharmain Belcastro 1 , Seongjin Seo 2 , MaximSokolov 1 . 1 Ophthalmology, West Virginia University, Morgantown,WV; 2 Ophthalmology and Visual Sciences, University of IowaCollege of Medicine, Iowa, IA.Purpose: Previously, we demonstrated that the development of therod outer segment, a sensory cilia responsible for light detection, iscrucially dependent on the normal function of the cytosolicchaperonin containing T-complex protein 1, CCT. Our goal here was©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyto identify the underlying molecular mechanism for such aphenotype.Methods: The activity of CCT in photoreceptors and cell culture wassuppressed by overexpressing a short splice isoform of phosducinlikeprotein, PhLPs. Cellular morphology was analyzed by lightmicroscopy and electron microscopy. Protein-protein interactions ofCCT were determined using an immunoprecipitation assay coupled toLC/MS/MS. Protein expression levels were studied byimmunofluorescence and Western blotting.Results: We identified several proteins that were severely downregulatedin response to CCT suppression and involved in outersegment morphogenesis. Those included Bardet-Biedl Syndromeproteins 2, 5 and 7, essential subunits of the cilia BBSome complex,and oxysterol-binding protein 2 (OSPB2), implicated in non-vesicularlipid trafficking. Interestingly, the reduction of rhodopsin andperipherin appeared to be secondary event, as both proteins were stilltargeted to the outer segment remnants.Conclusions: Our data provide evidence that CCT may potentially beinvolved in posttranslational processing of BBS 2, BBS5, BBS7, andOSPB2, which thus underlines its crucial importance in outersegment morphogenesis.Commercial Relationships: Satyabrata Sinha, None;Marycharmain Belcastro, None; Seongjin Seo, None; MaximSokolov, NoneSupport: NIH Grant EY019665, RPBProgram Number: 1228Presentation Time: 9:45 AM - 10:00 AMThe farnesylated small GTPase RAB28 is mutated in autosomalrecessive cone-rod dystrophySusanne Roosing 1, 2 , Klaus Rohrschneider 3 , Avigail Beryozkin 4 ,Nicole Weisschuh 5 , Susanne Kohl 5 , Bernd Wissinger 5 , Eyal Banin 4 ,Frans P. Cremers 1, 2 , Anneke I. Den Hollander 1, 6 . 1 Human Genetics,Radboud University Nijmegen Medical Centre, Nijmegen,Netherlands; 2 Nijmegen Centre for Molecular Life sciences,Nijmegen, Netherlands; 3 Department of Ophthalmology, Universityof Heidelberg, Heidelberg, Germany; 4 Department ofOphthalmology, Hadassah-Hebrew University Medical Center,Jerusalem, Israel; 5 Molecular Genetics Laboratory, Institute forOphthalmic Research, Centre for Ophthalmology, Tuebingen,Germany; 6 Department of Ophthalmology, Radboud UniversityNijmegen Medical Centre, Nijmegen, Netherlands.Purpose: The majority of genetic causes for autosomal recessive (ar)cone-rod dystrophy (CRD) are currently unknown. Therefore, weemployed a combined approach of homozygosity mapping andexome sequencing to identify new genes for arCRD.Methods: In a German arCRD family with three affected siblings,homozygosity mapping was performed using Affymetrix 250K SNPmicroarrays. DNA samples of two affected individuals underwentexome sequencing using Agilent’s SureSelect Human All Exon v.2Kit on a SOLiD4 sequencing platform. Sanger sequencing of theRAB28 gene was performed in 617 additional unrelated individualswith CRD or cone dystrophy, and in families with conspicuouslylarge homozygous regions assessed in SNP data available through theEuropean Retinal Disease Consortium. Identified mutations werescreened in ethnically matched controls. Ophthalmic examinationsincluded ERG, perimetry, OCT, FAF, and fundus photography.Results: Exome sequencing revealed a homozygous nonsensemutation in RAB28 (c.565C>T;p.Q189*) in all three affectedindividuals of the German arCRD family. In addition, a homozygousnonsense mutation (c.409C>T;p.R137*) was identified in twoaffected members of a consanguineous arCRD family of MoroccanJewish ancestry. Both mutations were not identified in 176 and 118ethnically matched controls, respectively. The five affectedindividuals of both families presented with hyperpigmentation in themacula, progressive loss of the visual acuity, atrophy of the retinalpigment epithelium, and severely reduced cone and rod responses onthe electroretinogram.Conclusions: RAB28 encodes a member of the Rab subfamily of theRAS-related small GTPases. Alternative RNA splicing yields threepredicted protein isoforms with alternative C-termini, which are alltruncated by the nonsense mutations identified in this study. Opposedto other Rab GTPases which are generally geranylgeranylated,RAB28 is predicted to be farnesylated. Interestingly, mutations inAIPL1, encoding a chaperone of farnesylated proteins, werepreviously found in individuals with ar Leber congenital amaurosisand autosomal dominant CRD. Analogous to the function of otherRAB family members, RAB28 might be involved in ciliogenesisand/or opsin transport in photoreceptor cells. This study reveals acrucial role for RAB28 in photoreceptor function, and suggests thatmutations in other Rab proteins may also be associated with retinaldystrophies.Commercial Relationships: Susanne Roosing, None; KlausRohrschneider, None; Avigail Beryozkin, None; NicoleWeisschuh, None; Susanne Kohl, None; Bernd Wissinger, None;Eyal Banin, None; Frans P. Cremers, None; Anneke I. DenHollander, NoneSupport: Foundation Fighting Blindness USA (grants BR-GE-0510-04890RAD and C-GE-0811-0545-RAD01)235 Ocular Disease Expression, Proteomics, Biomarkers, andPolymorphismsMonday, May 06, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 1574-1595/D0001-D0022Organizing Section: Biochemistry/Molecular BiologyProgram Number: 1574 Poster Board Number: D0001Presentation Time: 8:30 AM - 10:15 AMNew Potential Biomarker of Neurofibromatosis Type I,discovered with Multi-Spectral Imaging (MSI) of the RetinalPigment Epithelium (RPE) and ChoroidDorothy Hitchmoth 4, 1 , Jerome Sherman 2, 3 . 1 Surgery, Department ofVeterans Affairs, White River Junction, VT; 2 Clinical Science,SUNY College of Optometry, New York, NY; 3 SUNY Eye Institute,State University of New York, New York, NY; 4 Affiliate ResidencyPrograms, New England College of Optometry, Boston, MA.Purpose: The purpose of our research was to document the natureand type of choroidal lesions that were detected with a novelcommercially available imaging device.Methods: A retrospective analysis of the examination records andimages of 210 consecutive patients who were imaged with theAnnidis RHA. Ten sequential images were obtained, each with adifferent color LED with a unique spectrum. Seven of 10 of theseLEDs were in the red and infra-red part of the spectrum. Visiblelesions greater than .5 DD on one or more of the 10 images wereidentified. Patient records of those with such documented lesionswere evaluated in detail. Ultra-widefield (UWF) color images(Optos) were available in each case. In select cases, UWF FA, AF,Spectralis OCT, AF, FA, Topcon OCT and color images were alsoavailable.Results: 1)There was one patient with approximately 15 lesions ineach eye with corresponding dermatological lesions consistent withNeurofibromatosis Type I (NFI-1). The lesions were highly definedby MSI with red and infra-red LEDs but were invisible on©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
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