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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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Effects of OPTNE50K-GFP expression on Tf uptake, total TfR level, and TfR distribution in RPE cells.(A) Fluorescent images of RPE cells transfected with pOPTNE50K-GFP for 16 h and incubated with TR-Tf for 15 min. The transfected cells are in green (Aa) and the internalized TR-Tf is seen in red (Ab). Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in GFP (green filled circles)- and OPTNE50K-GFP (red triangles)-expressing cells. Fluorescence intensity of TR-Tf in the cells was measured at various time points. *, P<0.0001 compared to GFP controls. (C) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNE50K-GFP (lane 2) were immunoblotted with anti-TfR (Ca), anti-optineurin (OPTN) (Cb), and anti-GAPDH (Cc) antibodies. The expression level of 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNE50K-GFP band were detected. The GAPDH level was used to control protein loading. (D) RPE cells transfected with pOPTNE50K-GFP (Da, green) were immunostained with anti-TfR (Db, red). Merged image (Dc) shows colocalization between OPTNE50K-GFP and TfR in yellow.
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pone-0011547-g005: Effects of OPTNE50K-GFP expression on Tf uptake, total TfR level, and TfR distribution in RPE cells.(A) Fluorescent images of RPE cells transfected with pOPTNE50K-GFP for 16 h and incubated with TR-Tf for 15 min. The transfected cells are in green (Aa) and the internalized TR-Tf is seen in red (Ab). Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in GFP (green filled circles)- and OPTNE50K-GFP (red triangles)-expressing cells. Fluorescence intensity of TR-Tf in the cells was measured at various time points. *, P<0.0001 compared to GFP controls. (C) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNE50K-GFP (lane 2) were immunoblotted with anti-TfR (Ca), anti-optineurin (OPTN) (Cb), and anti-GAPDH (Cc) antibodies. The expression level of 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNE50K-GFP band were detected. The GAPDH level was used to control protein loading. (D) RPE cells transfected with pOPTNE50K-GFP (Da, green) were immunostained with anti-TfR (Db, red). Merged image (Dc) shows colocalization between OPTNE50K-GFP and TfR in yellow.

Mentions: We have shown that overexpression of a disease-causing mutant optineurin, OPTNE50K, induced foci formation more prominently than that seen in the wild type [25]. Here, experiments were carried out to evaluate whether E50K expression affects the Tf uptake. As seen in Fig. 5A, the TR-Tf accumulation within 15 min in OPTNE50K-GFP-expressing cells was much reduced compared to that in the neighboring nontransfected and GFP control (data not shown) cells. Quantitative analyses (Fig. 5B) showed that the TR-Tf uptake was significantly (P<0.0001) reduced and the reduction was more pronounced in cells expressing OPTNE50K-GFP (approximately 70–80% reduction) than OPTNWT-GFP (approximately 30–40% reduction, Fig. 1B).


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)

Effects of OPTNE50K-GFP expression on Tf uptake, total TfR level, and TfR distribution in RPE cells.(A) Fluorescent images of RPE cells transfected with pOPTNE50K-GFP for 16 h and incubated with TR-Tf for 15 min. The transfected cells are in green (Aa) and the internalized TR-Tf is seen in red (Ab). Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in GFP (green filled circles)- and OPTNE50K-GFP (red triangles)-expressing cells. Fluorescence intensity of TR-Tf in the cells was measured at various time points. *, P<0.0001 compared to GFP controls. (C) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNE50K-GFP (lane 2) were immunoblotted with anti-TfR (Ca), anti-optineurin (OPTN) (Cb), and anti-GAPDH (Cc) antibodies. The expression level of 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNE50K-GFP band were detected. The GAPDH level was used to control protein loading. (D) RPE cells transfected with pOPTNE50K-GFP (Da, green) were immunostained with anti-TfR (Db, red). Merged image (Dc) shows colocalization between OPTNE50K-GFP and TfR in yellow.
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pone-0011547-g005: Effects of OPTNE50K-GFP expression on Tf uptake, total TfR level, and TfR distribution in RPE cells.(A) Fluorescent images of RPE cells transfected with pOPTNE50K-GFP for 16 h and incubated with TR-Tf for 15 min. The transfected cells are in green (Aa) and the internalized TR-Tf is seen in red (Ab). Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in GFP (green filled circles)- and OPTNE50K-GFP (red triangles)-expressing cells. Fluorescence intensity of TR-Tf in the cells was measured at various time points. *, P<0.0001 compared to GFP controls. (C) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNE50K-GFP (lane 2) were immunoblotted with anti-TfR (Ca), anti-optineurin (OPTN) (Cb), and anti-GAPDH (Cc) antibodies. The expression level of 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNE50K-GFP band were detected. The GAPDH level was used to control protein loading. (D) RPE cells transfected with pOPTNE50K-GFP (Da, green) were immunostained with anti-TfR (Db, red). Merged image (Dc) shows colocalization between OPTNE50K-GFP and TfR in yellow.
Mentions: We have shown that overexpression of a disease-causing mutant optineurin, OPTNE50K, induced foci formation more prominently than that seen in the wild type [25]. Here, experiments were carried out to evaluate whether E50K expression affects the Tf uptake. As seen in Fig. 5A, the TR-Tf accumulation within 15 min in OPTNE50K-GFP-expressing cells was much reduced compared to that in the neighboring nontransfected and GFP control (data not shown) cells. Quantitative analyses (Fig. 5B) showed that the TR-Tf uptake was significantly (P<0.0001) reduced and the reduction was more pronounced in cells expressing OPTNE50K-GFP (approximately 70–80% reduction) than OPTNWT-GFP (approximately 30–40% reduction, Fig. 1B).

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