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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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Oligomerization of optineurin.A. Native blue gel electrophoresis. Total protein from RGC5 cells (30 µg) and that (15 µg) from RGC5 cells transfected for 20 h to express FLAG-optineurin (FLAG-OPTN) fusion protein were subjected to native blue gel electrophoresis and Western blotting. The membranes were probed either with anti-optineurin to detect the endogenous optineurin (left lane) or anti-FLAG to detect the forced expressed FLAG-OPTN fusion protein (right lane). A band at approximately 420 kDa was observed in both cases in the native gel. With the FLAG-OPTN, higher molecular weight bands which may represent the complex formed with other optineurin binding partners were additionally seen. B. Co-immunoprecipiation. RGC5 cells were co-transfected with pTarget-FLAG-OPTN and pOPTN-His. Cell lysates were immunoprecipitated with rabbit anti-His antibody, and the immunoprecipitated proteins were probed with anti-FLAG antibody under reducing conditions. An immuoreactive optineurin band (arrowhead) was detected in the anti-His (IP His, left lane) but not in the rabbit IgG (IP Control, right lane) pull down. Size markers are indicated.
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pone-0009168-g005: Oligomerization of optineurin.A. Native blue gel electrophoresis. Total protein from RGC5 cells (30 µg) and that (15 µg) from RGC5 cells transfected for 20 h to express FLAG-optineurin (FLAG-OPTN) fusion protein were subjected to native blue gel electrophoresis and Western blotting. The membranes were probed either with anti-optineurin to detect the endogenous optineurin (left lane) or anti-FLAG to detect the forced expressed FLAG-OPTN fusion protein (right lane). A band at approximately 420 kDa was observed in both cases in the native gel. With the FLAG-OPTN, higher molecular weight bands which may represent the complex formed with other optineurin binding partners were additionally seen. B. Co-immunoprecipiation. RGC5 cells were co-transfected with pTarget-FLAG-OPTN and pOPTN-His. Cell lysates were immunoprecipitated with rabbit anti-His antibody, and the immunoprecipitated proteins were probed with anti-FLAG antibody under reducing conditions. An immuoreactive optineurin band (arrowhead) was detected in the anti-His (IP His, left lane) but not in the rabbit IgG (IP Control, right lane) pull down. Size markers are indicated.

Mentions: The secondary structure prediction based on the amino acid sequence of both human and rat optineurin showed that it is rich in α-helical coils (∼68%) (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html and http://www.russell.embl.de/cgi-bin/coils-svr.pl), which supercoil to form protein-protein interaction coiled coil domains [25]. To examine the possibility that optineurin can form oligomers or complexes, lysates from RGC5 cells were run on native blue gels followed by Western blotting for the endogenous optineurin under non-reducing conditions. A single band was detected at approximately 420-kDa, mass equivalent of homo-hexamer, indicating that optineurin interacted with itself to form hexamers (Fig. 5A, left lane).


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)

Oligomerization of optineurin.A. Native blue gel electrophoresis. Total protein from RGC5 cells (30 µg) and that (15 µg) from RGC5 cells transfected for 20 h to express FLAG-optineurin (FLAG-OPTN) fusion protein were subjected to native blue gel electrophoresis and Western blotting. The membranes were probed either with anti-optineurin to detect the endogenous optineurin (left lane) or anti-FLAG to detect the forced expressed FLAG-OPTN fusion protein (right lane). A band at approximately 420 kDa was observed in both cases in the native gel. With the FLAG-OPTN, higher molecular weight bands which may represent the complex formed with other optineurin binding partners were additionally seen. B. Co-immunoprecipiation. RGC5 cells were co-transfected with pTarget-FLAG-OPTN and pOPTN-His. Cell lysates were immunoprecipitated with rabbit anti-His antibody, and the immunoprecipitated proteins were probed with anti-FLAG antibody under reducing conditions. An immuoreactive optineurin band (arrowhead) was detected in the anti-His (IP His, left lane) but not in the rabbit IgG (IP Control, right lane) pull down. Size markers are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820081&req=5

pone-0009168-g005: Oligomerization of optineurin.A. Native blue gel electrophoresis. Total protein from RGC5 cells (30 µg) and that (15 µg) from RGC5 cells transfected for 20 h to express FLAG-optineurin (FLAG-OPTN) fusion protein were subjected to native blue gel electrophoresis and Western blotting. The membranes were probed either with anti-optineurin to detect the endogenous optineurin (left lane) or anti-FLAG to detect the forced expressed FLAG-OPTN fusion protein (right lane). A band at approximately 420 kDa was observed in both cases in the native gel. With the FLAG-OPTN, higher molecular weight bands which may represent the complex formed with other optineurin binding partners were additionally seen. B. Co-immunoprecipiation. RGC5 cells were co-transfected with pTarget-FLAG-OPTN and pOPTN-His. Cell lysates were immunoprecipitated with rabbit anti-His antibody, and the immunoprecipitated proteins were probed with anti-FLAG antibody under reducing conditions. An immuoreactive optineurin band (arrowhead) was detected in the anti-His (IP His, left lane) but not in the rabbit IgG (IP Control, right lane) pull down. Size markers are indicated.
Mentions: The secondary structure prediction based on the amino acid sequence of both human and rat optineurin showed that it is rich in α-helical coils (∼68%) (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html and http://www.russell.embl.de/cgi-bin/coils-svr.pl), which supercoil to form protein-protein interaction coiled coil domains [25]. To examine the possibility that optineurin can form oligomers or complexes, lysates from RGC5 cells were run on native blue gels followed by Western blotting for the endogenous optineurin under non-reducing conditions. A single band was detected at approximately 420-kDa, mass equivalent of homo-hexamer, indicating that optineurin interacted with itself to form hexamers (Fig. 5A, left lane).

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