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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyDorie Miller, William Hansen Sandberg Found.(JDD, KL), USCProvost Undergrad. Research(KL)Program Number: 2453 Poster Board Number: D0058Presentation Time: 2:45 PM - 4:30 PMCharacterization and Post-Translational Modifications ofProline-Rich Protein 4 (PRR4) in Basal and Reflex TearProteome of HumanNatarajan Perumal, Sebastian Funke, Norbert Pfeiffer, Franz H.Grus. Experimental Ophthalmology, University Medical CenterMainz, Mainz, Germany.Purpose: It was previously reported that PRR4 is down-regulated inthe tears of dry eye patients. This study was initiated considering thelack of comprehensive characteristics profile of PRR4 in tears ofhealthy subjects and, to understand the physiological roles of PRR4in tears. Post-translational modifications and characterization ofPRR4 in basal and reflex tears were determined in the studypresented herein.Methods: Tear samples were collected employing capillary methodfor basal and reflex tears. Proteomics approaches based on 1-DE and2-DE combined with LC-ESI-LTQ-Orbitrap MS system wereemployed for characterization and determination of post-translationalmodifications of PRR4.Results: Identification and characterization of PRR4 in 2-DEutilizing LC-ESI-LTQ-Orbitrap MS yielded 10 PRR4 spots with vastpolymorphisms (molecular weight: 17 - 30 kDa, pI: 3.5 - 6.6). A newisoform designated as PRR4γ was also identified in addition to thetwo existing isoforms of PRR4. Further characterization of the PRR4isoforms identified multiple post-translational modifications, whichare methylation, oxidation and pyroglutamate formation. Label-freequantification analysis of basal and reflex tear proteome employing1-DE and LC-ESI-LTQ-Orbitrap MS demonstrated significantincrement of PRR4 for the first time in reflex tears. Correspondingly,the result also showed that more methylation, oxidation andpyroglutamate formation were found in reflex tears than in basaltears. Besides, reflex tear sample also showed significant incrementof prolactin, serum albumin, zinc-α-2-glycoprotein, haptoglobin,mesothelin and β-2-microglobulin and, decrement of Ig α-1 chain C,cystatin-S, clusterin, lipid transfer protein II and phospholipase A2compared to basal tears.Conclusions: The results of this study provide fundamental insightinto the complex characteristics of PRR4 in basal and reflex tears.These findings highlight that the multiple post-translationalmodifications of PRR4 isoforms may play crucial roles in the tearsystem and ocular surface of healthy people as well as in dry eyepatients.Commercial Relationships: Natarajan Perumal, None; SebastianFunke, None; Norbert Pfeiffer, Sensimed AG (F), Sensimed AG(R), MSD (F), MSD (R), Alcon (F), Allergan (F), Novartis (F),Novartis (R), Bayer (F), Heidelberg Engineering (F), Bausch&Lomb(F), Boehringer-Ingelheim (F), Carl Zeiss Meditech (F), Chibret (F),Nidek (F), Pfizer (F), Santen (F), Santen (R), Topcon (F), Ivantis Inc(F), Ivantis Inc (R); Franz H. Grus, NoneProgram Number: 2454 Poster Board Number: D0059Presentation Time: 2:45 PM - 4:30 PMSteric Volume Exclusion Leads to Size-dependent ProteinDistribution in Mouse Ciliary PhotoreceptorsMehdi Najafi 1, 2 , Peter D. Calvert 1, 2 . 1 Department of Ophthalmology,SUNY Upstate Medical University, Syracuse, NY; 2 SUNY EyeInstitute, Syracuse, NY.Purpose: We recently demonstrated that ‘steric volume exclusion’mediates size-dependent soluble protein distribution in Xenopuslaevis rod photoreceptors 1 . However, the large variation in themorphology of photoreceptors among species raises the question ofwhether the steric volume exclusion effect is significant for allphotoreceptors. We therefore examined the subcellular distributionsof soluble molecules of different sizes in mouse photoreceptors usinglive cell confocal imaging.Methods: Microslices of retina from dark-adapted mice expressingEGFP in rod cells under the NRL promoter were prepared underinfrared illumination, placed in an imaging chamber containingmouse ringer solution, and imaged using our custom confocalmicroscope. Distribution of EGFP in rods was compared to that ofthe small fluorescent probe Calcein introduced into WT retinas thatwere similarly prepared. Corrections were made to account for thefact that mouse rods are below the resolution limit of confocalmicrocopy.Results: The distribution of Calcein significantly differed from thatof EGFP in mouse photoreceptors. The ratio of the meanfluorescence in the outer segment to that in the myoid region(F OS /F MY ) was 0.91±0.03 in photoreceptors containing Calcein, anddropped to 0.44 ±0.06 in EGFP expressing rods. A size-dependentdistribution was evident in other areas of the cell as well. WhileCalcein uniformly filled the nucleus, EGFP was excluded from thecentral areas of this compartment where heterochromatin is located.Conclusions: Our results indicate that steric volume exclusiongoverns protein distribution in mammalian photoreceptors in muchthe same manner as in Xenopus. The lower F OS /F MY ratio in EGFPexpressing rods compared to Calcein demonstrates a size-dependentexclusion from the outer segment due to the higher packing densityof disk membranes in this compartment. We project a molecular sizecut-off of ~4.0nm for protein access to the outer segment in mousewhich is consistent with our previous estimation in Xenopusphotoreceptors (4.2 nm). Our experiments indicate that despitesignificant variation in photoreceptor morphology among species,steric volume exclusion remains a general mechanism for regulationof protein access to the ciliary outersegment compartment.1. Najafi et al. PNAS, 012 Jan 3;109(1):203-8Commercial Relationships: Mehdi Najafi, None; Peter D. Calvert,NoneSupport: NIH Grant EY018421, Research to Prevent BlindnessProgram Number: 2455 Poster Board Number: D0060Presentation Time: 2:45 PM - 4:30 PMRefined estimates of rhodopsin deactivation kinetics from mouserods lacking rhodopsin phosphorylation or Arr1 bindingOwen P. Gross 1, 2 , Edward N. Pugh 2, 4 , Marie E. Burns 2, 3 . 1 VollumInstitute, Oregon Health & Science University, Portland, OR; 2 Centerfor Neuroscience, UC Davis, Davis, CA; 3 Ophthalmology & VisionScience, UC Davis, Davis, CA; 4 Cell Biology and Human Anatomy,UC Davis, Davis, CA.Purpose: Deactivation of rhodopsin (R*) requires multiplephosphorylation of its COOH-terminal serine and threonine residuesby Grk1, followed by the binding of arrestin-1 (Arr1). Single cellrecordings from rods that under-express Arr1 have suggested that R*lifetime is normally brief and dictated largely by phosphorylation(Gross and Burns, 2010). Here, we apply a recently developed modelof phototransduction to single photon responses (SPRs) of rodslacking either phosphorylation or Arr1 binding, providing newquantitative estimates of the in situ kinetics of R* deactivation.Methods: Suction electrode recordings from intact rod outersegments were performed as previously described. Measured SPRswere compared to simulations produced by a spatiotemporalphototransduction model (Gross et al 2012a). The model input was atime series representing R* activity, generated assuming a Markov©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 Biologydeactivation process in which Arr1 binds with high affinity after threephosphorylations.Results: The Grk1-/- SPR is approximately a step function (Chen etal., 1999), whose ~2-fold larger amplitude constrains the maximal R*activity in the context of the downstream model, and whose 3-4 smean duration sets the upper limit for the rate of Arr1 binding tounphosphorylated R*. In contrast, Arr1-/- SPRs exhibit peakamplitudes similar to wild-type SPRs and have long-lasting tailcurrents with amplitudes ~50% of the peak (e.g. Xu et al., 1997).Simulated SPRs revealed that this tail current can be explained by arelatively small fraction (~5%) of maximal R* activity that persistsafter extensive phosphorylation of R*. In order to achieve this lowlevel of R* activity on the time scale of the SPR, phosphorylation ofR* must proceed at a maximal, initial rate of 100/s.Conclusions: The deduced R* deactivation kinetics predict that 3-4G protein/PDE (G*-E*) complexes are active during the plateau tailphase of the Arr1-/- SPR, a number that corresponds to ~50% of theG*-E* level at the peak of the wild-type SPR. In contrast, ~ 60 G*-E* complexes are predicted for the Grk1-/- SPR. The grossdisproportionality between the number of G*-E* complexes and theresponse plateau amplitudes reveals the consequences of cascadesaturation and the disproportionate role of calcium feedback to cGMPsynthesis on larger responses (Gross et al., 2012b).Commercial Relationships: Owen P. Gross, None; Edward N.Pugh, None; Marie E. Burns, NoneSupport: NIH Grant R01-EY014047 (MEB)Program Number: 2456 Poster Board Number: D0061Presentation Time: 2:45 PM - 4:30 PMRobust self-association of arrestin-1 is a neuroprotectivemechanismEugenia V. Gurevich 1 , Xiufeng Song 1 , Jungwon Seo 1 , FaizaBaameur 1 , Sergey A. Vishnivetskiy 1 , Qiuyan Chen 1 , Miyeon Kim 2 ,Jeannie Chen 3 , Wayne L. Hubbell 2 , Vsevolod V. Gurevich 1 .1 Pharmacology, Vanderbilt University, Nashville, TN; 2 University ofCalifornia Los Angeles, Los Angeles, CA; 3 University of SouthernCalifornia, Los Angeles, CA.Purpose: To determine the biological role of arrestin-1 selfassociation.Methods: Retinal morphology and the health of rod photoreceptorsin mice of different ages expressing wild type (WT) arrestin-1 andmutant with impaired self-association was compared.Results: Arrestin-1 binds light-activated phosphorhodopsin andensures timely signal shutoff. Arrestin-1 self-association is conservedin at least three mammalian species, but its biological role remainsobscure. We found that high expression of arrestin-1 mutant withimpaired oligomerization, but not of WT arrestin-1 that robustlyoligomerizes, results in progressive rod degeneration via apoptosis.Synaptic terminals of rods are adversely affected earlier than othercell compartments. Dark rearing does not prevent mutant-inducedcell death, ruling out the role of arrestin-rhodopsin complexes. WTarrestin-1 co-expressed with the mutant delays retinal degeneration,likely by forming mixed oligomers.Conclusions: Monomeric arrestin-1 is cytotoxic, and it likelyacquired propensity to oligomerize to reduce the concentration of themonomer in the cell. This finding suggests that cones express thebulk of their arrestin complement (~98%) in the form of arrestin-1because cone-specific arrestin-4 is naturally oligomerization-deficientand therefore likely cytotoxic. The results also suggest that arrestin-1binding to microtubules largely serves to minimize free monomer inthe cell body, rather than to localize arrestin-1 away from the outersegment in the dark.Commercial Relationships: Eugenia V. Gurevich, None; XiufengSong, None; Jungwon Seo, None; Faiza Baameur, None; Sergey A.Vishnivetskiy, None; Qiuyan Chen, None; Miyeon Kim, None;Jeannie Chen, None; Wayne L. Hubbell, None; Vsevolod V.Gurevich, NoneSupport: NIH grants, EY011500, GM077561, GM081756 (VVG),NS45117 and NS065868 (EVG), EY05216 and the Jules SteinProfessorship Endowment (WLH), EY012155 (JC)Program Number: 2457 Poster Board Number: D0062Presentation Time: 2:45 PM - 4:30 PMAdaptive acceleration in mouse rods is mediated by slowfeedback via guanylate cyclase activating proteinsMarie E. Burns 1, 2 , Owen P. Gross 1 , Claudia M. Krispel 3, 4 . 1 Centerfor Neuroscience, Univ of California-Davis, Davis, CA; 2 CellBiology and Human Anatomy, Univ of California-Davis, Davis, CA;3 Ophthalmology & Vision Science, Univ of California-Davis, Davis,CA; 4 Wilmer Eye Institute, Johns Hopkins Hospital, Baltimore, MD.Purpose: In retinal photoreceptors, adaptation to steady backgroundlight produces several specific changes in phototransductionbiochemistry and the incremental flash response. A long-lasting formof adaptation termed “adaptive acceleration” (AA) has been found toshorten responses for many tens of seconds following the offset ofsaturating light, but the mechanism and functional significance of thisform of adaptation has been unknown.Methods: Suction electrodes were used to record the outer segmentmembrane current of individual rods of mice before, during, and afteran adapting stimulus (500 nm light) that varied both in intensity andduration. Adaptive acceleration was probed using brief (10 ms)flashes of light of fixed intensity. The dominant time constant ofrecovery was probed using saturating flashes whose intensity varied2-8 fold in the period immediately before and after the adapting light,and by measuring the time that these flash responses remained insaturation.Results: As described previously, AA was easily induced usingsteady light that suppressed the rod’s circulating current. Theinduction of AA required extended periods of response saturation,with an onset time constant of 35 s, and faded with a time constant of~80 s. Analysis of a sequence of saturating flash responsesimmediately after the adapting light offset revealed that, contrary toan earlier report, AA was not associated with the shortening of thedominant time constant of recovery, indicating that it is not caused byacceleration of GTPase activity. Indeed, rods that overexpressed theRGS9 complex or which lacked several other targets for adaptation,including recoverin, showed the normal induction and offset featuresof AA. However, AA was completely abolished in the rods of micelacking guanylate cyclase activating proteins, or GCAPs.Furthermore, AA improved the flicker response properties of wildtyperods, but not in rods lacking GCAPs.Conclusions: GCAPs have previously been described to play a rolein setting the single photon response amplitude and in preventingsaturation in steady light (Mendez et al., 2001; Burns et al., 2002;Gross et al 2012). Our findings in the current study suggest thatGCAPs also mediate a longer-lasting change in guanylate cyclaseactivity that improves the temporal resolution of the rods following abright steady light.Commercial Relationships: Marie E. Burns, None; Owen P.Gross, None; Claudia M. Krispel, NoneSupport: NIH Grant R01-EY014047 (MEB)Program Number: 2458 Poster Board Number: D0063Presentation Time: 2:45 PM - 4:30 PM©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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