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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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Self binding of optineurin mutants and their interactions with Rab8 and TfR.(A) Self binding of wild type optineurin and mutants. RPE cells were co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT, pOPTNE50K-His + pTarget-FLAG-OPTNE50K, or pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cell lysates were immunoprecipitated (IP) with polyclonal anti-His. The IP eluates were immunoblotted (IB) with monoclonal anti-FLAG or monoclonal anti-His. The anticipated molecular weight of the proteins detected by both antibodies was 75 kDa. No protein band was detected when rabbit IgG was used for immunoprecipitation (data not shown). (B) Binding of wild type optineurin and mutants with endogenous Rab8. Inducible RGC5 cells were treated with doxycycline for 16 h to express OPTNWT-GFP, OPTNE50K-GFP and OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with polyclonal anti-GFP. The IP eluates were immunoblotted (IB) with monoclonal anti-Rab8 or polyclonal anti-GFP. The intensity ratio between the detected 27-kDa-Rab8 band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown). (C) Binding of wild type optineurin and mutants with endogenous TfR. Inducible stable cell lines were induced by doxycycline to express OPTNWT-GFP, OPTNE50K-GFP or OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-GFP antibody. The IP eluates were immunoblotted (IB) with a monoclonal anti-TfR antibody or anti-GFP. The intensity ratio between the 95-kDa TfR band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown).
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pone-0011547-g007: Self binding of optineurin mutants and their interactions with Rab8 and TfR.(A) Self binding of wild type optineurin and mutants. RPE cells were co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT, pOPTNE50K-His + pTarget-FLAG-OPTNE50K, or pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cell lysates were immunoprecipitated (IP) with polyclonal anti-His. The IP eluates were immunoblotted (IB) with monoclonal anti-FLAG or monoclonal anti-His. The anticipated molecular weight of the proteins detected by both antibodies was 75 kDa. No protein band was detected when rabbit IgG was used for immunoprecipitation (data not shown). (B) Binding of wild type optineurin and mutants with endogenous Rab8. Inducible RGC5 cells were treated with doxycycline for 16 h to express OPTNWT-GFP, OPTNE50K-GFP and OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with polyclonal anti-GFP. The IP eluates were immunoblotted (IB) with monoclonal anti-Rab8 or polyclonal anti-GFP. The intensity ratio between the detected 27-kDa-Rab8 band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown). (C) Binding of wild type optineurin and mutants with endogenous TfR. Inducible stable cell lines were induced by doxycycline to express OPTNWT-GFP, OPTNE50K-GFP or OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-GFP antibody. The IP eluates were immunoblotted (IB) with a monoclonal anti-TfR antibody or anti-GFP. The intensity ratio between the 95-kDa TfR band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown).

Mentions: Our laboratory has previously reported that wild type optineurin interacts with itself to form homo-hexamer as well as with its binding partners to form supermolecular complexes [36]. The foci formation is presumably related to these complex formations. To determine whether a point mutation of optineurin at amino acid residue 50 from Glu to Lys or residue 157 from Leu to Ala would change its ability to self interact, RPE cells were co-transfected with pOPTNE50K-His + pTarget-FLAG-OPTNE50K and pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cells were also co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT as a positive control. The lysates were immunoprecipitated with a polyclonal anti-His antibody and the IP eluates were immunoblotted with a monoclonal anti-FLAG antibody (Fig. 7A, top panel). The anticipated molecular weight of the proteins detected by anti-FLAG antibody was 75 kDa. The membrane was also immunoblotted with anti-His monoclonal antibody to verify that IP was properly performed (Fig. 7A, lower panel). The results revealed that both E50K and L157A mutants were still capable of binding to themselves and the binding was comparable to that of the wild type optineurin. Native blue gel electrophoresis performed further detected formation of the 420 kDa homo-hexamer by the wild type and the two optineurin mutants (data not shown).


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)

Self binding of optineurin mutants and their interactions with Rab8 and TfR.(A) Self binding of wild type optineurin and mutants. RPE cells were co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT, pOPTNE50K-His + pTarget-FLAG-OPTNE50K, or pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cell lysates were immunoprecipitated (IP) with polyclonal anti-His. The IP eluates were immunoblotted (IB) with monoclonal anti-FLAG or monoclonal anti-His. The anticipated molecular weight of the proteins detected by both antibodies was 75 kDa. No protein band was detected when rabbit IgG was used for immunoprecipitation (data not shown). (B) Binding of wild type optineurin and mutants with endogenous Rab8. Inducible RGC5 cells were treated with doxycycline for 16 h to express OPTNWT-GFP, OPTNE50K-GFP and OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with polyclonal anti-GFP. The IP eluates were immunoblotted (IB) with monoclonal anti-Rab8 or polyclonal anti-GFP. The intensity ratio between the detected 27-kDa-Rab8 band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown). (C) Binding of wild type optineurin and mutants with endogenous TfR. Inducible stable cell lines were induced by doxycycline to express OPTNWT-GFP, OPTNE50K-GFP or OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-GFP antibody. The IP eluates were immunoblotted (IB) with a monoclonal anti-TfR antibody or anti-GFP. The intensity ratio between the 95-kDa TfR band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown).
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Related In: Results  -  Collection

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pone-0011547-g007: Self binding of optineurin mutants and their interactions with Rab8 and TfR.(A) Self binding of wild type optineurin and mutants. RPE cells were co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT, pOPTNE50K-His + pTarget-FLAG-OPTNE50K, or pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cell lysates were immunoprecipitated (IP) with polyclonal anti-His. The IP eluates were immunoblotted (IB) with monoclonal anti-FLAG or monoclonal anti-His. The anticipated molecular weight of the proteins detected by both antibodies was 75 kDa. No protein band was detected when rabbit IgG was used for immunoprecipitation (data not shown). (B) Binding of wild type optineurin and mutants with endogenous Rab8. Inducible RGC5 cells were treated with doxycycline for 16 h to express OPTNWT-GFP, OPTNE50K-GFP and OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with polyclonal anti-GFP. The IP eluates were immunoblotted (IB) with monoclonal anti-Rab8 or polyclonal anti-GFP. The intensity ratio between the detected 27-kDa-Rab8 band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown). (C) Binding of wild type optineurin and mutants with endogenous TfR. Inducible stable cell lines were induced by doxycycline to express OPTNWT-GFP, OPTNE50K-GFP or OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-GFP antibody. The IP eluates were immunoblotted (IB) with a monoclonal anti-TfR antibody or anti-GFP. The intensity ratio between the 95-kDa TfR band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown).
Mentions: Our laboratory has previously reported that wild type optineurin interacts with itself to form homo-hexamer as well as with its binding partners to form supermolecular complexes [36]. The foci formation is presumably related to these complex formations. To determine whether a point mutation of optineurin at amino acid residue 50 from Glu to Lys or residue 157 from Leu to Ala would change its ability to self interact, RPE cells were co-transfected with pOPTNE50K-His + pTarget-FLAG-OPTNE50K and pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cells were also co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT as a positive control. The lysates were immunoprecipitated with a polyclonal anti-His antibody and the IP eluates were immunoblotted with a monoclonal anti-FLAG antibody (Fig. 7A, top panel). The anticipated molecular weight of the proteins detected by anti-FLAG antibody was 75 kDa. The membrane was also immunoblotted with anti-His monoclonal antibody to verify that IP was properly performed (Fig. 7A, lower panel). The results revealed that both E50K and L157A mutants were still capable of binding to themselves and the binding was comparable to that of the wild type optineurin. Native blue gel electrophoresis performed further detected formation of the 420 kDa homo-hexamer by the wild type and the two optineurin mutants (data not shown).

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