Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyeyes. A single antibody (Cell Signaling Technology) was able tospecifically detect recombinant p15INK4B by Western blot and inpositive skin squamous cells. This antibody labeled cell nuclei in theretinal inner nuclear and ganglion cell layers and glial columns in theoptic nerve.Conclusions: Analysis of the distribution of gene products from ornear the 9p21 gene region in the human eye indicated that restinglevels of P16INK4A/P14ARF were too low to detect but that bothMTAP and p15INK4B were present in different cells. This workprovides a framework for future studies that will seek to explore howthese gene products may influence the pathogenesis of POAG.Commercial Relationships: John P. Wood, None; Glyn Chidlow,None; Robert J. Casson, None; Shiwani Sharma, None; KathrynP. Burdon, None; Jamie E. Craig, NoneSupport: NH&MRC Grant ID APP1031347Program Number: 1607 Poster Board Number: D0034Presentation Time: 8:30 AM - 10:15 AMAnalysis of the covalent high molecular weight protein complexcontaining optineurin and its relationship with glaucomaJie Gao 1, 2 , Masafumi Ohtsubo 1 , Yoshihiro Hotta 2 , ShinseiMinoshima 1 . 1 Department of Photomedical Genomics, Basic MedicalPhotonics Laboratory, Medical Photonics Research Center,Hamamatsu University School of Medicine, Hamamatsu, Japan;2 Department of Ophthalmology, Hamamatsu University School ofMedicine, Hamamatsu, Japan.Purpose: We are analyzing the function of optineurin (OPTN) toelucidate the onset mechanism of glaucoma. It was reported that thephysiologically existing form of OPTN in cultured cells is oligomer,which is dissociated into monomers when detected by westernblotting (WB) using denatured gels under reducing condition. Werecently found an OPTN-containing high molecular weight complex(HMC) induced by H 2 O 2 treatment of cells using the same detectionmethod and named it covalent HMC. The aim of this study is toanalyze the covalent HMC and its relationship with glaucomapathogenesis.Methods: Plasmids to express wild-type (Wt) and a series of mutantOPTNs were constructed and transfected into HeLaS3 or NIH3T3cells. The mutants include the glaucoma causative ones, H26D,E50K, M98K, H486R, and R545Q, as well as a designed mutationD474N which was reported to abolish the binding with ubiquitin (Ub)and a truncated mutant LcUBD (lacking Ub-Binding Domain). TheHMC formed in the cells transfected with these constructswith/without stimuli was detected by WB using denatured gels underreducing condition. Dual-luciferase reporter assay was applied formeasuring TNFα-induced NF-κB activity (TINA) to examine if theinhibitory effect of OPTN on TINA is abolished by LcUBD andD474N.Results: 1. The covalent HMC induced by H 2 O 2 treatment of cellswas probably the covalent trimer of OPTN (cOPTN 3 ) according tothe estimated molecular weight from the WB results; 2. Theformation of cOPTN 3 was detected in not only Wt but also all of thepoint mutants including D474N when cells were stimulated withH 2 O 2 ; 3. Only in the case of E50K, the formation of cOPTN 3 wasinduced without H 2 O 2 addition; 4. cOPTN 3 was not detected inLcUBD with/without H 2 O 2 treatment; 5. TNFα did not induce theformation of cOPTN 3 .Conclusions: 1. Our results suggested that physiologically existingnon-covalent OPTN oligomer is cross-linked each other with somecovalent bond under oxidative environment with unknownmechanism. 2. E50K is a distinct mutation which possibly producesthe similar intracellular condition as H 2 O 2 treatment, suggesting apossible relationship of E50K with the severity of glaucomaphenotype. 3. The UBD is necessary for cOPTN 3 formation, but thesite essential for Ub binding seems unnecessary. 4. cOPTN 3 is notinvolved in TNFα-induced NF-κB signaling pathway.Commercial Relationships: Jie Gao, None; Masafumi Ohtsubo,None; Yoshihiro Hotta, None; Shinsei Minoshima, NoneProgram Number: 1608 Poster Board Number: D0035Presentation Time: 8:30 AM - 10:15 AMPost-transcriptional regulation of γ-synuclein expression and itsrole in glaucomatous alterationsAndrei Surguchov 1, 2 , Irina G. Surgucheva 1, 2 . 1 Research, RetinalBiology Lab, VAMCKC, Kansas City, MO; 2 Neurology, KansasUniversity, Kansas City, KS.Purpose: Glaucoma is related to the accumulation of aggregated andmisfolded proteins. γ-Synuclein (γ-Syn) accumulates in the retina andin the optic nerve astrocytes in large spheroidal structures in patientswith glaucoma and in animal models forming protease resistantintracellular inclusions. Defects in the regulation of γ-Syn expressionare responsible for the accumulation of such pathological deposits.microRNAs (miRNAs) are small, non-coding RNAs, that regulategene expression through their targets in the 3'untranslated regions ofmRNAs. Here we investigate the mechanism of post-transcriptionalregulation of γ-Syn expression by miRNAs.Methods: A 275 bp fragment was generated by PCR using human γ-Syn clone (long form, LF). LF and modified forms of this fragmentwith deleted putative miRNAs targets were inserted in a Luc reportervector and Luc activity was tested after transient transfection.Expression of miRNAs was carried out using “BLOCK-iT Pol II miRRNAi Expression Vector”. miRNAs were quantified using qRT-PCRwith TaqMan probes.Results: First we analyzed γ-Syn 3’-UTR by bioinformatics approachand identified putative targets for the following miRNAs: miR-103,107, 424, 497, 4437 and 4674 and 4722. The insertion of LFdownstream of the LUC gene caused a 51% reduction of LUCactivity, confirming the presence of effective targets for miRNAs inthis fragment. Deletion of targets for miR-103 and miR-107 from LFdoes not change the activity, although according to qRT-PCR datathese miRNAs are present in tested cell cultures. The effect oftruncated form (TF) of the 3’-UTR in which we have modifiedtargets for miR-4437 and miR-4674 on LUC activity was different: itdid not change LUC activity, while the deletion of miR-103 targetsfrom this TF increases the activity. To further investigate the effect ofmiR4437 and miR4674 on γ-Syn expression we performedexpression of these two miRNAs in SK-BR3 cells. According toWestern blotting data, expression of miR4437 caused a 69% andmiR4674 a 76% reduction of endogenous γ-Syn expression. On theother hand, in stable clones overexpressing γ-Syn no significanteffect of miRNAs on γ-Syn expression was found.Conclusions: 3’-UTR contains targets for miRNAs which are usedfor the regulation of γ-Syn expression. This mechanism of regulationis cell-specific and depends on the level of γ-Syn and miRNAsexpression.Commercial Relationships: Andrei Surguchov, None; Irina G.Surgucheva, NoneSupport: VA Merit Review, The Glaucoma FoundationProgram Number: 1609 Poster Board Number: D0036Presentation Time: 8:30 AM - 10:15 AMSoluble guanylate cyclase: an emerging therapeutic target inopen angle glaucomaEmmanuel S. Buys 1 , Yu-Chieh Ko 2 , Clemens Alt 3 , Haiyan Gong 4 ,Peter Brouckaert 5 , Janey L. Wiggs 6 , Meredith S. Gregory-Ksander 2, 6 ,Louis R. Pasquale 6, 7 , Kenneth D. Bloch 1, 8 , Bruce R. Ksander 2, 6 .©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. 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