Impairment of protein trafficking upon overexpression and mutation of optineurin.

PloS One
BumChan ParkBeatrice Y J T Yue

Abstract

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. 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 t...Continue Reading

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Apr 1, 2014·SpringerPlus·Sanja TurturroHongyu Ying
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Methods Mentioned

BETA
GTPase
co-immunoprecipitation
transfection
electrophoresis
immunoprecipitation
pull down
pull downs
Fluorescence
PCR
Antibody Capture

Software Mentioned

Metamorph
PROSITE
Leica confocal

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