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Impairment of protein trafficking upon overexpression and mutation of optineurin.

Park B, Ying H, Shen X, Park JS, Qiu Y, Shyam R, Yue BY - PLoS ONE (2010)

Bottom Line: A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells.Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation.

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 characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.

Methodology/principal findings: Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.

Conclusions/significance: The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.

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Overexpression of wild type and E50K optineurin, but not L157A mutant, alters the surface TfR level in RPE cells.Cells were transfected to express GFP, as well as wild type (WT)-, E50K-, and L157A-optineurin-GFP. Surface TfR was detected by Western blotting with anti-TfR after surface protein biotinylation and isolation of biotinylated proteins. The level of surface TfR was normalized to that of a well known cell surface protein, β1-integrin. The ratios of TfR to β1-integrin are presented. Ctl, nontransfected control RPE cells.
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pone-0011547-g008: Overexpression of wild type and E50K optineurin, but not L157A mutant, alters the surface TfR level in RPE cells.Cells were transfected to express GFP, as well as wild type (WT)-, E50K-, and L157A-optineurin-GFP. Surface TfR was detected by Western blotting with anti-TfR after surface protein biotinylation and isolation of biotinylated proteins. The level of surface TfR was normalized to that of a well known cell surface protein, β1-integrin. The ratios of TfR to β1-integrin are presented. Ctl, nontransfected control RPE cells.

Mentions: To provide further evidence that the sequestration of TfR by the optineurin foci may be an important factor in blocking of the Tf uptake, the level of surface TfR was determined. The surface proteins in transfected RPE cells were labeled with non-permeable sulfo-NHS-SS-biotin reagent as described previously [38]. Results (Fig. 8) showed that relative to a well-known surface protein, β1-integrin, the surface TfR level in OPTNWT-GFP and OPTNE50K-GFP overexpressing cells was markedly decreased than that in GFP controls. Transfection with pOPTNL157A-GFP, on the other hand, did not lower the TfR level on the cell surface.


Impairment of protein trafficking upon overexpression and mutation of optineurin.

Park B, Ying H, Shen X, Park JS, Qiu Y, Shyam R, Yue BY - PLoS ONE (2010)

Overexpression of wild type and E50K optineurin, but not L157A mutant, alters the surface TfR level in RPE cells.Cells were transfected to express GFP, as well as wild type (WT)-, E50K-, and L157A-optineurin-GFP. Surface TfR was detected by Western blotting with anti-TfR after surface protein biotinylation and isolation of biotinylated proteins. The level of surface TfR was normalized to that of a well known cell surface protein, β1-integrin. The ratios of TfR to β1-integrin are presented. Ctl, nontransfected control RPE cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902519&req=5

pone-0011547-g008: Overexpression of wild type and E50K optineurin, but not L157A mutant, alters the surface TfR level in RPE cells.Cells were transfected to express GFP, as well as wild type (WT)-, E50K-, and L157A-optineurin-GFP. Surface TfR was detected by Western blotting with anti-TfR after surface protein biotinylation and isolation of biotinylated proteins. The level of surface TfR was normalized to that of a well known cell surface protein, β1-integrin. The ratios of TfR to β1-integrin are presented. Ctl, nontransfected control RPE cells.
Mentions: To provide further evidence that the sequestration of TfR by the optineurin foci may be an important factor in blocking of the Tf uptake, the level of surface TfR was determined. The surface proteins in transfected RPE cells were labeled with non-permeable sulfo-NHS-SS-biotin reagent as described previously [38]. Results (Fig. 8) showed that relative to a well-known surface protein, β1-integrin, the surface TfR level in OPTNWT-GFP and OPTNE50K-GFP overexpressing cells was markedly decreased than that in GFP controls. Transfection with pOPTNL157A-GFP, on the other hand, did not lower the TfR level on the cell surface.

Bottom Line: A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells.Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation.

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 characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.

Methodology/principal findings: Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.

Conclusions/significance: The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.

Show MeSH
Related in: MedlinePlus