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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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Rescue of Tf uptake by co-transfection with TfR but not with myosin VI and Rab8 constructs in RPE cells.(A) Fluorescent images of RPE cells. The cells transfected with pOPTNWT-GFP (OPTN) alone, or co-transfected with pOPTNWT-GFP and pMyoVI-EGFP (OPTN + HM6), pRab8Q67L-EGFP (OPTN + Rab8), or pTfR-EGFP (OPTN + TfR) were incubated with Texas Red-Tf (TR-Tf) for 15 min. The single or co-transfected cells are in green and the internalized TR-Tf is seen in red. The yellow staining in perinuclear regions (bottom panel, merged) indicated interaction and colocalization between the expressed TfR-GFP and the internalized TR-Tf. Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in nontransfected and single- or co-transfected RPE cells. Note that co-transfection with myosin VI and Rab8 constructs had little effect, while co-expression of TfR restored the Tf uptake to the nontransfected control level in OPTNWT-GFP-expressing cells. Data are expressed as mean ± SD. Experiments are repeated at least three times, yielding similar results. *, P<0.001 compared to nontransfected cells.
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pone-0011547-g003: Rescue of Tf uptake by co-transfection with TfR but not with myosin VI and Rab8 constructs in RPE cells.(A) Fluorescent images of RPE cells. The cells transfected with pOPTNWT-GFP (OPTN) alone, or co-transfected with pOPTNWT-GFP and pMyoVI-EGFP (OPTN + HM6), pRab8Q67L-EGFP (OPTN + Rab8), or pTfR-EGFP (OPTN + TfR) were incubated with Texas Red-Tf (TR-Tf) for 15 min. The single or co-transfected cells are in green and the internalized TR-Tf is seen in red. The yellow staining in perinuclear regions (bottom panel, merged) indicated interaction and colocalization between the expressed TfR-GFP and the internalized TR-Tf. Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in nontransfected and single- or co-transfected RPE cells. Note that co-transfection with myosin VI and Rab8 constructs had little effect, while co-expression of TfR restored the Tf uptake to the nontransfected control level in OPTNWT-GFP-expressing cells. Data are expressed as mean ± SD. Experiments are repeated at least three times, yielding similar results. *, P<0.001 compared to nontransfected cells.

Mentions: To determine whether the impairment of Tf uptake could be rescued by optineurin binding proteins myosin VI, Rab8, or TfR, RPE (Fig. 3) and RGC5 (data not shown) cells were co-transfected with pOPTNWT-GFP along with pMyoVI-EGFP, pRab8Q67L-EGFP, or pTfR-EGFP [36] for 16 h. They were then incubated with TR-Tf and the Tf uptake was quantified. It was found that co-transfection with myosin VI and Rab8 constructs (Fig. 3A) had little effect, while co-expression of TfR expression plasmid (Fig. 3A) restored the Tf uptake to the nontransfected control level in OPTNWT-GFP-expressing cells (Fig. 3B). The yellow staining in perinuclear regions (Fig. 3A, bottom panel, merged) indicated interaction and colocalization between the expressed TfR-GFP and the internalized TR-Tf.


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)

Rescue of Tf uptake by co-transfection with TfR but not with myosin VI and Rab8 constructs in RPE cells.(A) Fluorescent images of RPE cells. The cells transfected with pOPTNWT-GFP (OPTN) alone, or co-transfected with pOPTNWT-GFP and pMyoVI-EGFP (OPTN + HM6), pRab8Q67L-EGFP (OPTN + Rab8), or pTfR-EGFP (OPTN + TfR) were incubated with Texas Red-Tf (TR-Tf) for 15 min. The single or co-transfected cells are in green and the internalized TR-Tf is seen in red. The yellow staining in perinuclear regions (bottom panel, merged) indicated interaction and colocalization between the expressed TfR-GFP and the internalized TR-Tf. Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in nontransfected and single- or co-transfected RPE cells. Note that co-transfection with myosin VI and Rab8 constructs had little effect, while co-expression of TfR restored the Tf uptake to the nontransfected control level in OPTNWT-GFP-expressing cells. Data are expressed as mean ± SD. Experiments are repeated at least three times, yielding similar results. *, P<0.001 compared to nontransfected cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902519&req=5

pone-0011547-g003: Rescue of Tf uptake by co-transfection with TfR but not with myosin VI and Rab8 constructs in RPE cells.(A) Fluorescent images of RPE cells. The cells transfected with pOPTNWT-GFP (OPTN) alone, or co-transfected with pOPTNWT-GFP and pMyoVI-EGFP (OPTN + HM6), pRab8Q67L-EGFP (OPTN + Rab8), or pTfR-EGFP (OPTN + TfR) were incubated with Texas Red-Tf (TR-Tf) for 15 min. The single or co-transfected cells are in green and the internalized TR-Tf is seen in red. The yellow staining in perinuclear regions (bottom panel, merged) indicated interaction and colocalization between the expressed TfR-GFP and the internalized TR-Tf. Scale bar, 20 µm. (B) Quantification of TR-Tf uptake in nontransfected and single- or co-transfected RPE cells. Note that co-transfection with myosin VI and Rab8 constructs had little effect, while co-expression of TfR restored the Tf uptake to the nontransfected control level in OPTNWT-GFP-expressing cells. Data are expressed as mean ± SD. Experiments are repeated at least three times, yielding similar results. *, P<0.001 compared to nontransfected cells.
Mentions: To determine whether the impairment of Tf uptake could be rescued by optineurin binding proteins myosin VI, Rab8, or TfR, RPE (Fig. 3) and RGC5 (data not shown) cells were co-transfected with pOPTNWT-GFP along with pMyoVI-EGFP, pRab8Q67L-EGFP, or pTfR-EGFP [36] for 16 h. They were then incubated with TR-Tf and the Tf uptake was quantified. It was found that co-transfection with myosin VI and Rab8 constructs (Fig. 3A) had little effect, while co-expression of TfR expression plasmid (Fig. 3A) restored the Tf uptake to the nontransfected control level in OPTNWT-GFP-expressing cells (Fig. 3B). The yellow staining in perinuclear regions (Fig. 3A, bottom panel, merged) indicated interaction and colocalization between the expressed TfR-GFP and the internalized TR-Tf.

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