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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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Related in: MedlinePlus

Cellular localization of OPTNL157A-GFP and effects of OPTNL157A-GFP expression on Tf uptake, total TfR level, and TfR distribution.(A) RPE cells were transfected with pEGFP-N1 (GFP mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c) for 16 h. Representative confocal images shown in Aa and Ab depict two major patterns of OPTNL157A distribution. Scale bar, 20 µm. (B) The number of foci in at least 60 RPE transfectants was counted and the average number of foci per transfectant is shown. Data (mean ± SD) presented are representative from three independent experiments. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (C) Human TM cells were transfected pEGFP-N1 (mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c). Two patterns of OPTNL157A distribution are seen in a and b. Scale bar, 20 µm. (D) The number of foci in TM transfectants was counted. The average number of foci per transfectant (mean ± SD) is presented. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (E) Fluorescent images of OPTNL157A-GFP-expressing RPE cells after internalization of TR-Tf for 15 min. The transfected cells are in green (Ea) and the internalized TR-Tf is shown in red (Eb). Scale bar, 20 µm. (F) Quantification of TR-Tf uptake in OPTNL157A-transfected RPE cells. Fluorescence intensity of TR-Tf, determined as in Fig. 1B, is expressed as percentage relative to that of the 15-min uptake in GFP-expressing controls. Data (mean ± SD) presented are representative of three independent experiments. (G) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNL157A-GFP (lane 2) were immunoblotted with anti-TfR (Ga), anti-optineurin (OPTN) (Gb), and anti-GAPDH (Gc, used to control protein loading) antibodies. Total level of the 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNL157A-GFP band were detected. (H) RPE cells transfected with pOPTNL157A-GFP (Ha, green) were immunostained with anti-TfR (Hb, red). The diffuse distribution of OPTNL157A-GFP overlapped with that of TfR in yellow in the merged image (Hc).
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pone-0011547-g006: Cellular localization of OPTNL157A-GFP and effects of OPTNL157A-GFP expression on Tf uptake, total TfR level, and TfR distribution.(A) RPE cells were transfected with pEGFP-N1 (GFP mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c) for 16 h. Representative confocal images shown in Aa and Ab depict two major patterns of OPTNL157A distribution. Scale bar, 20 µm. (B) The number of foci in at least 60 RPE transfectants was counted and the average number of foci per transfectant is shown. Data (mean ± SD) presented are representative from three independent experiments. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (C) Human TM cells were transfected pEGFP-N1 (mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c). Two patterns of OPTNL157A distribution are seen in a and b. Scale bar, 20 µm. (D) The number of foci in TM transfectants was counted. The average number of foci per transfectant (mean ± SD) is presented. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (E) Fluorescent images of OPTNL157A-GFP-expressing RPE cells after internalization of TR-Tf for 15 min. The transfected cells are in green (Ea) and the internalized TR-Tf is shown in red (Eb). Scale bar, 20 µm. (F) Quantification of TR-Tf uptake in OPTNL157A-transfected RPE cells. Fluorescence intensity of TR-Tf, determined as in Fig. 1B, is expressed as percentage relative to that of the 15-min uptake in GFP-expressing controls. Data (mean ± SD) presented are representative of three independent experiments. (G) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNL157A-GFP (lane 2) were immunoblotted with anti-TfR (Ga), anti-optineurin (OPTN) (Gb), and anti-GAPDH (Gc, used to control protein loading) antibodies. Total level of the 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNL157A-GFP band were detected. (H) RPE cells transfected with pOPTNL157A-GFP (Ha, green) were immunostained with anti-TfR (Hb, red). The diffuse distribution of OPTNL157A-GFP overlapped with that of TfR in yellow in the merged image (Hc).

Mentions: Leucine zipper motif is known to be sites for protein-protein interactions. Optineurin protein contains one leucine zipper domain (amino acid residues 143–164) at the N-terminus and Rab8 has been suggested to interact with optineurin through the N-terminal region (amino acid residues 141–209) that includes the leucine zipper domain [12], [19]–[22], [37]. To obliterate this domain, we introduced a point mutation by in vitro mutagenesis to substitute Leu157 to Ala (L157A) in the optineurin sequence. OPTNL157A construct in pEGFP vector (pOPTNL157A-GFP) was made. When transfected, two patterns of OPTNL157A-GFP distribution were noted in human RPE (Fig. 6A), TM (Fig. 6C), and RGC5 (data not shown) cells. In about 60–70% of transfected cells, foci formation was hardly seen and the OPTNL157A-GFP fusion protein distributed diffusely throughout the cytoplasm (Fig. 6Aa and 6Ca). Foci were observed in the remaining cells but they were not concentrated in the perinuclear area (Fig. 6Ab and 6Cb). Compared to those seen in wild type optineurin-expressing cells, the OPTNL157A foci appeared to be fewer in number, smaller in size, and more dispersed into the cytoplasm. Quantitative counting confirmed the microscopic observations that cells transfected with pOPTNL157A-GFP had on average a lower number of foci compared with those transfected with pOPTNWT-GFP (10.3±11 versus 50.3 ±1,9 in RPE cells, P<0.0001, Fig. 6B; 11.5±2.3 versus 64.6±2.1 in TM cells, P<0.0001, Fig. 6D).


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)

Cellular localization of OPTNL157A-GFP and effects of OPTNL157A-GFP expression on Tf uptake, total TfR level, and TfR distribution.(A) RPE cells were transfected with pEGFP-N1 (GFP mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c) for 16 h. Representative confocal images shown in Aa and Ab depict two major patterns of OPTNL157A distribution. Scale bar, 20 µm. (B) The number of foci in at least 60 RPE transfectants was counted and the average number of foci per transfectant is shown. Data (mean ± SD) presented are representative from three independent experiments. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (C) Human TM cells were transfected pEGFP-N1 (mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c). Two patterns of OPTNL157A distribution are seen in a and b. Scale bar, 20 µm. (D) The number of foci in TM transfectants was counted. The average number of foci per transfectant (mean ± SD) is presented. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (E) Fluorescent images of OPTNL157A-GFP-expressing RPE cells after internalization of TR-Tf for 15 min. The transfected cells are in green (Ea) and the internalized TR-Tf is shown in red (Eb). Scale bar, 20 µm. (F) Quantification of TR-Tf uptake in OPTNL157A-transfected RPE cells. Fluorescence intensity of TR-Tf, determined as in Fig. 1B, is expressed as percentage relative to that of the 15-min uptake in GFP-expressing controls. Data (mean ± SD) presented are representative of three independent experiments. (G) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNL157A-GFP (lane 2) were immunoblotted with anti-TfR (Ga), anti-optineurin (OPTN) (Gb), and anti-GAPDH (Gc, used to control protein loading) antibodies. Total level of the 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNL157A-GFP band were detected. (H) RPE cells transfected with pOPTNL157A-GFP (Ha, green) were immunostained with anti-TfR (Hb, red). The diffuse distribution of OPTNL157A-GFP overlapped with that of TfR in yellow in the merged image (Hc).
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getmorefigures.php?uid=PMC2902519&req=5

pone-0011547-g006: Cellular localization of OPTNL157A-GFP and effects of OPTNL157A-GFP expression on Tf uptake, total TfR level, and TfR distribution.(A) RPE cells were transfected with pEGFP-N1 (GFP mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c) for 16 h. Representative confocal images shown in Aa and Ab depict two major patterns of OPTNL157A distribution. Scale bar, 20 µm. (B) The number of foci in at least 60 RPE transfectants was counted and the average number of foci per transfectant is shown. Data (mean ± SD) presented are representative from three independent experiments. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (C) Human TM cells were transfected pEGFP-N1 (mock control, not shown), pOPTNL157A-GFP (a and b) or pOPTNWT-GFP (c). Two patterns of OPTNL157A distribution are seen in a and b. Scale bar, 20 µm. (D) The number of foci in TM transfectants was counted. The average number of foci per transfectant (mean ± SD) is presented. *, P<0.0001 compared to OPTNWT-GFP-expressing cells. (E) Fluorescent images of OPTNL157A-GFP-expressing RPE cells after internalization of TR-Tf for 15 min. The transfected cells are in green (Ea) and the internalized TR-Tf is shown in red (Eb). Scale bar, 20 µm. (F) Quantification of TR-Tf uptake in OPTNL157A-transfected RPE cells. Fluorescence intensity of TR-Tf, determined as in Fig. 1B, is expressed as percentage relative to that of the 15-min uptake in GFP-expressing controls. Data (mean ± SD) presented are representative of three independent experiments. (G) Lysates (50 µg total protein) from RPE cells transfected with pEGFP-N1 (lane 1) or pOPTNL157A-GFP (lane 2) were immunoblotted with anti-TfR (Ga), anti-optineurin (OPTN) (Gb), and anti-GAPDH (Gc, used to control protein loading) antibodies. Total level of the 95-kDa TfR was similar in lanes 1 and 2. A 74-kDa endogenous optineurin band and a 100-kDa OPTNL157A-GFP band were detected. (H) RPE cells transfected with pOPTNL157A-GFP (Ha, green) were immunostained with anti-TfR (Hb, red). The diffuse distribution of OPTNL157A-GFP overlapped with that of TfR in yellow in the merged image (Hc).
Mentions: Leucine zipper motif is known to be sites for protein-protein interactions. Optineurin protein contains one leucine zipper domain (amino acid residues 143–164) at the N-terminus and Rab8 has been suggested to interact with optineurin through the N-terminal region (amino acid residues 141–209) that includes the leucine zipper domain [12], [19]–[22], [37]. To obliterate this domain, we introduced a point mutation by in vitro mutagenesis to substitute Leu157 to Ala (L157A) in the optineurin sequence. OPTNL157A construct in pEGFP vector (pOPTNL157A-GFP) was made. When transfected, two patterns of OPTNL157A-GFP distribution were noted in human RPE (Fig. 6A), TM (Fig. 6C), and RGC5 (data not shown) cells. In about 60–70% of transfected cells, foci formation was hardly seen and the OPTNL157A-GFP fusion protein distributed diffusely throughout the cytoplasm (Fig. 6Aa and 6Ca). Foci were observed in the remaining cells but they were not concentrated in the perinuclear area (Fig. 6Ab and 6Cb). Compared to those seen in wild type optineurin-expressing cells, the OPTNL157A foci appeared to be fewer in number, smaller in size, and more dispersed into the cytoplasm. Quantitative counting confirmed the microscopic observations that cells transfected with pOPTNL157A-GFP had on average a lower number of foci compared with those transfected with pOPTNWT-GFP (10.3±11 versus 50.3 ±1,9 in RPE cells, P<0.0001, Fig. 6B; 11.5±2.3 versus 64.6±2.1 in TM cells, P<0.0001, Fig. 6D).

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