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Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene.

Ying H, Shen X, Park B, Yue BY - PLoS ONE (2010)

Bottom Line: It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein.Treatment of nocadazole resulted in dispersion of the optineurin foci.The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin.

Methodology/principal findings: Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin-green fluorescence protein (GFP) fusion protein formed punctate structures termed "foci" in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time.

Conclusions/significance: The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP-expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.

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Distribution of optineurin foci is microtubule dependent.RGC5 (A) and RPE (B) cells expressing optineurin-GFP (OPTN-GFP) were untreated (Control) or treated with 10 µM nocodazole for 30 min. The cells were washed thoroughly to remove nocodazole (Washout) and allowed to recover for 1 h. The optineurin-GFP foci appeared in green and the microtubule network was visualized by immunostaining with anti-α-tubulin in red. The microtubule was disrupted upon nocodazole treatment and the optineurin foci were dispersed from perinuclear region to distribute evenly in the cytoplasm. Upon nocadazole removal, the microtubule network was restored and the foci returned to the perinuclear area. Scale bar, 10 µm.
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pone-0009168-g004: Distribution of optineurin foci is microtubule dependent.RGC5 (A) and RPE (B) cells expressing optineurin-GFP (OPTN-GFP) were untreated (Control) or treated with 10 µM nocodazole for 30 min. The cells were washed thoroughly to remove nocodazole (Washout) and allowed to recover for 1 h. The optineurin-GFP foci appeared in green and the microtubule network was visualized by immunostaining with anti-α-tubulin in red. The microtubule was disrupted upon nocodazole treatment and the optineurin foci were dispersed from perinuclear region to distribute evenly in the cytoplasm. Upon nocadazole removal, the microtubule network was restored and the foci returned to the perinuclear area. Scale bar, 10 µm.

Mentions: The long distance movement of optineurin foci (Video S2) suggested that their distribution might depend on the microtubule network. pOPTN-EGFP-transfected RGC5 (Fig. 4A) and RPE (Fig. 4B) cells were treated with nocadazole, an agent that interferes with microtubule polymerization. Disruption of the network by nocadazole was confirmed by α-tubulin staining (Fig. 4) and under this condition, the optineurin foci were dispersed from the perinuclear region to distribute evenly in the cytoplasm. When nocadazole was removed, the microtubule was recovered and the optineurin foci were again clustered around the perinuclear region, similar to that seen in the untreated controls.


Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene.

Ying H, Shen X, Park B, Yue BY - PLoS ONE (2010)

Distribution of optineurin foci is microtubule dependent.RGC5 (A) and RPE (B) cells expressing optineurin-GFP (OPTN-GFP) were untreated (Control) or treated with 10 µM nocodazole for 30 min. The cells were washed thoroughly to remove nocodazole (Washout) and allowed to recover for 1 h. The optineurin-GFP foci appeared in green and the microtubule network was visualized by immunostaining with anti-α-tubulin in red. The microtubule was disrupted upon nocodazole treatment and the optineurin foci were dispersed from perinuclear region to distribute evenly in the cytoplasm. Upon nocadazole removal, the microtubule network was restored and the foci returned to the perinuclear area. Scale bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820081&req=5

pone-0009168-g004: Distribution of optineurin foci is microtubule dependent.RGC5 (A) and RPE (B) cells expressing optineurin-GFP (OPTN-GFP) were untreated (Control) or treated with 10 µM nocodazole for 30 min. The cells were washed thoroughly to remove nocodazole (Washout) and allowed to recover for 1 h. The optineurin-GFP foci appeared in green and the microtubule network was visualized by immunostaining with anti-α-tubulin in red. The microtubule was disrupted upon nocodazole treatment and the optineurin foci were dispersed from perinuclear region to distribute evenly in the cytoplasm. Upon nocadazole removal, the microtubule network was restored and the foci returned to the perinuclear area. Scale bar, 10 µm.
Mentions: The long distance movement of optineurin foci (Video S2) suggested that their distribution might depend on the microtubule network. pOPTN-EGFP-transfected RGC5 (Fig. 4A) and RPE (Fig. 4B) cells were treated with nocadazole, an agent that interferes with microtubule polymerization. Disruption of the network by nocadazole was confirmed by α-tubulin staining (Fig. 4) and under this condition, the optineurin foci were dispersed from the perinuclear region to distribute evenly in the cytoplasm. When nocadazole was removed, the microtubule was recovered and the optineurin foci were again clustered around the perinuclear region, similar to that seen in the untreated controls.

Bottom Line: It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein.Treatment of nocadazole resulted in dispersion of the optineurin foci.The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin.

Methodology/principal findings: Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin-green fluorescence protein (GFP) fusion protein formed punctate structures termed "foci" in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time.

Conclusions/significance: The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP-expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.

Show MeSH
Related in: MedlinePlus