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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 optineurin overexpression on the total level and distribution of the endogenous TfR in RPE cells.(A )Lysates (50 µg total protein) from cells transfected with pEGFP-N1 (lane 1) or pOPTNWT-GFP (lane 2) were immunoblotted with anti-TfR (Aa), anti-optineurin (OPTN) (Ab), and anti-GAPDH (Ac) antibodies. The expression level of 95-kDa TfR was not reduced by OPTNWT-GFP overexpression. Anti-OPTN detected the 74-kDa endogenous OPTN band in both lanes and a 100-kDa OPTNWT-GFP band in lane 2. Anti-GAPDH that detected a 37-kDa band, was used to control protein loading. (B) Distribution of TfR in GFP- and OPTNWT-GFP-expressing cells. RPE cells transfected with pEGFP-N1 (Ba-Bc, green) and pOPTNWT-GFP (Bd-Bf, green) were immunostained with anti-TfR (Bb, Bc, Be, Bf, red). Merged images are shown in Bc and Bf. Colocalization between OPTNWT-GFP and TfR is seen in yellow (Bf).
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pone-0011547-g004: Effects of optineurin overexpression on the total level and distribution of the endogenous TfR in RPE cells.(A )Lysates (50 µg total protein) from cells transfected with pEGFP-N1 (lane 1) or pOPTNWT-GFP (lane 2) were immunoblotted with anti-TfR (Aa), anti-optineurin (OPTN) (Ab), and anti-GAPDH (Ac) antibodies. The expression level of 95-kDa TfR was not reduced by OPTNWT-GFP overexpression. Anti-OPTN detected the 74-kDa endogenous OPTN band in both lanes and a 100-kDa OPTNWT-GFP band in lane 2. Anti-GAPDH that detected a 37-kDa band, was used to control protein loading. (B) Distribution of TfR in GFP- and OPTNWT-GFP-expressing cells. RPE cells transfected with pEGFP-N1 (Ba-Bc, green) and pOPTNWT-GFP (Bd-Bf, green) were immunostained with anti-TfR (Bb, Bc, Be, Bf, red). Merged images are shown in Bc and Bf. Colocalization between OPTNWT-GFP and TfR is seen in yellow (Bf).

Mentions: The TfR protein level in pEGFP-N1 (mock control)- or pOPTNWT-GFP-transfected RPE cells was examined by Western blotting. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level was used as a protein loading control. In addition, the level of the endogenous optineurin and the OPTNWT-GFP fusion protein was also determined to confirm that the cells were indeed transfected. Results in Fig. 4A showed that the TfR level in cells expressing OPTNWT-GFP was similar to that of the mock control. The reduction of Tf uptake in pOPTNWT-GFP-transfected cells was thus unrelated to the expression level of total TfR.


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 optineurin overexpression on the total level and distribution of the endogenous TfR in RPE cells.(A )Lysates (50 µg total protein) from cells transfected with pEGFP-N1 (lane 1) or pOPTNWT-GFP (lane 2) were immunoblotted with anti-TfR (Aa), anti-optineurin (OPTN) (Ab), and anti-GAPDH (Ac) antibodies. The expression level of 95-kDa TfR was not reduced by OPTNWT-GFP overexpression. Anti-OPTN detected the 74-kDa endogenous OPTN band in both lanes and a 100-kDa OPTNWT-GFP band in lane 2. Anti-GAPDH that detected a 37-kDa band, was used to control protein loading. (B) Distribution of TfR in GFP- and OPTNWT-GFP-expressing cells. RPE cells transfected with pEGFP-N1 (Ba-Bc, green) and pOPTNWT-GFP (Bd-Bf, green) were immunostained with anti-TfR (Bb, Bc, Be, Bf, red). Merged images are shown in Bc and Bf. Colocalization between OPTNWT-GFP and TfR is seen in yellow (Bf).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902519&req=5

pone-0011547-g004: Effects of optineurin overexpression on the total level and distribution of the endogenous TfR in RPE cells.(A )Lysates (50 µg total protein) from cells transfected with pEGFP-N1 (lane 1) or pOPTNWT-GFP (lane 2) were immunoblotted with anti-TfR (Aa), anti-optineurin (OPTN) (Ab), and anti-GAPDH (Ac) antibodies. The expression level of 95-kDa TfR was not reduced by OPTNWT-GFP overexpression. Anti-OPTN detected the 74-kDa endogenous OPTN band in both lanes and a 100-kDa OPTNWT-GFP band in lane 2. Anti-GAPDH that detected a 37-kDa band, was used to control protein loading. (B) Distribution of TfR in GFP- and OPTNWT-GFP-expressing cells. RPE cells transfected with pEGFP-N1 (Ba-Bc, green) and pOPTNWT-GFP (Bd-Bf, green) were immunostained with anti-TfR (Bb, Bc, Be, Bf, red). Merged images are shown in Bc and Bf. Colocalization between OPTNWT-GFP and TfR is seen in yellow (Bf).
Mentions: The TfR protein level in pEGFP-N1 (mock control)- or pOPTNWT-GFP-transfected RPE cells was examined by Western blotting. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level was used as a protein loading control. In addition, the level of the endogenous optineurin and the OPTNWT-GFP fusion protein was also determined to confirm that the cells were indeed transfected. Results in Fig. 4A showed that the TfR level in cells expressing OPTNWT-GFP was similar to that of the mock control. The reduction of Tf uptake in pOPTNWT-GFP-transfected cells was thus unrelated to the expression level of total TfR.

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