Impairment of protein trafficking upon overexpression and mutation of optineurin.
Impairment of protein trafficking upon overexpression and mutation of optineurin.
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DOI：
10.1371/journal.pone.0011547
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发表时间：
2010-07-12
期刊：
影响因子：
3.7
通讯作者：
Yue BY
中科院分区：
文献类型：
--
作者：
Park B;Ying H;Shen X;Park JS;Qiu Y;Shyam R;Yue BY
青光眼是一种以视网膜神经节细胞(RGC)和轴突进行性丧失为特征的主要致盲疾病。Optineurin是目前已发现的候选基因之一。据报道，Glu50到Lys(E50K)的突变与一种更进展和更严重的疾病有关。Optineurin与Rab8、肌球蛋白VI和转铁蛋白受体(TFR)相互作用，被推测在蛋白质运输中发挥作用。在这里，我们确定了视神经磷酸酶过表达和E50K突变是否以及如何影响转铁蛋白(Tf)的内化，转铁蛋白被广泛用作受体介导的内吞作用的标志。人视网膜色素上皮(RPE)和大鼠RGC5细胞高表达野生型视神经磷酸酶(Optineurin)，与德州红TF孵育，检测Tf摄取。转染后在核周区域形成颗粒状结构或斑点。此外，在转基因细胞中还观察到转铁蛋白摄取的损害。与野生型的过表达相比，E50K突变产生了更多的病灶形成和更明显的TF摄取缺陷。与TFR共转染，而不是Rab8或肌球蛋白VI，构建挽救了视神经磷酸酶抑制效应，提示TFR是参与运输表型的因素。野生型和E50K Optineurin的强制表达使TFR与病灶共定位。表面生物素化实验表明，TFR的表面水平也降低了，可能导致Tf摄取受阻。一个Leu157到Ala(L157A)的非相应突变，其病灶形成和TFR结合显著减少，具有正常的TFR分布、正常的表面TFR水平和正常的TF内化。本研究表明，野生型视神经磷酸酶过表达导致RPE和RGC5细胞的TF摄取受损。这种表型与视神经磷酸酶与转铁蛋白受体的相互作用有关。我们的结果进一步表明，E50K比野生型Optineurin产生更显著的影响，因此是一种功能获得突变。蛋白转运缺陷可能是E50K突变患者青光眼病理发生的基础之一。
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 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. 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 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. 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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