Nagabhushana Ananthamurthy, Chalasani Madhavi L, Jain Nishant, Radha Vegesna, Rangaraj Nandini, Balasubramanian Dorairajan, Swarup Ghanshyam
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India.
BMC Cell Biol. 2010 Jan 19;11:4. doi: 10.1186/1471-2121-11-4.
Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-kappaB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.
We show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.
Our results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.
视紫质是一种多功能蛋白，参与多种功能，如从高尔基体到质膜的囊泡运输、核因子κB调节、信号转导和基因表达。视紫质突变与青光眼相关，青光眼是一种导致失明的神经退行性眼病。遗传证据表明，E50K（谷氨酸50突变为赖氨酸）是视紫质的主要致病突变。然而，视紫质突变引起的功能改变尚不清楚。在此，我们分析了视紫质在内吞再循环中的作用以及E50K突变体对这一过程的影响。
我们发现，视紫质的敲低会损害转铁蛋白受体向近核区域的运输。泛素结合结构域中的一个点突变（D474N）消除了视紫质在再循环内体中的定位以及与转铁蛋白受体的相互作用。视紫质泛素结合结构域的功能对于转铁蛋白向近核区域的运输也是必需的。一种致病突变E50K会损害转铁蛋白受体的内吞再循环，表现为再循环内体增大、E50K囊泡动力学减慢以及表达E50K的细胞对转铁蛋白的摄取减少。E50K突变体导致的这种运输受损需要其泛素结合结构域的功能。与野生型视紫质相比，E50K视紫质与转铁蛋白受体和Rab8的相互作用及共定位增强。E50K突变体的共表达降低了Rab8囊泡的速度。这些结果表明，E50K突变体影响Rab8介导的转铁蛋白受体运输。
我们的结果表明，视紫质调节转铁蛋白受体向近核区域的内吞运输。E50K突变体由于与Rab8和转铁蛋白受体的相互作用改变，损害了再循环内体的运输。这些结果也对视紫质E50K突变导致青光眼的发病机制具有启示意义，因为内吞再循环对于维持体内平衡至关重要。
