Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin

Abstract

BackgroundGlaucoma 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 Glu50 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 FindingsHuman 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 Leu157 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/SignificanceThe 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.

Optineurin mediates negative regulation of Rab8 function by TBC1D17, a GTPase activating protein

“…Activated Rab8 is known to form a complex with TfR, as shown by coimmunoprecipitation (Nagabhushana et al, 2010;Park et al, 2010). This is supported by preferential colocalisation of activated Rab8 with TfR (supplementary material Fig.…”

“…1H,I). Previously, it has been shown that optineurin is present in vesicles positive for TfR (Nagabhushana et al, 2010;Park et al, 2010). Hence, we examined the distribution of TfR in the cells co-expressing optineurin and TBC1D17.…”

“…Previous studies have shown that the E50K mutant of optineurin impairs endocytic trafficking of TfR, resulting in accumulation of TfR in vesicular structures in the cytoplasm (Nagabhushana et al, 2010;Park et al, 2010). This defective trafficking is possibly due to an altered interaction of the E50K mutant with Rab8 and also with TfR.…”

“…Optineurin preferentially interacts with activated form of Rab8 (Hattula and Peranen, 2000) and, like Rab8, is involved in regulating endocytic trafficking of transferrin and TfR (Hattula et al, 2006;Nagabhushana et al, 2010;Park et al, 2010). It has been reported that TBC1D17 (also known as FLJ12168) does not show direct interaction with Rab8 in a yeast two-hybrid assay (Itoh et al, 2006).…”

Toward an integrative view of Optineurin functions

“…17,67 Overexpression of Optn E50K also leads to impairment of transferrin uptake in human retinal pigment epithelial cells and in RGCs, indicating a defect in protein trafficking. 68 Thr210. [57][58][59] Recently, it has been shown that Optn plays a critical role in the inactivation of Plk1 during mitosis.…”