Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin.pdf (2.4 MB)

Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin

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posted on 27.05.2011 by BumChan Park, Hongyu Ying, Xiang Shen, Jeong-Seok Park, Ye Qiu, Rajalekshmy Shyam, Beatrice Y. J. T. Yue
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.

Funding

This work was supported by Grants EY018828, EY005628, and EY003890 (to B.Y.J.T.Y.) and Core Grant EY01792 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland; and by a grant from the American Health Assistance Foundation, Clarksburg, Maryland.

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Publisher Statement

© 2010 Park et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The original source for this publication is at the Public Library of Science. DOI:10.1371/journal.pone.0011547.

Publisher

Public Library of Science

Language

en_US

issn

1932-6203

Issue date

12/07/2010

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