Department of Pharmacology, Brain Science & Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Republic of Korea.
J Neurosci Res. 2018 Mar;96(3):407-415. doi: 10.1002/jnr.24143. Epub 2017 Aug 26.
Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN-mediated autophagy is a complex process involving many autophagy-regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large-scale yeast-human two-hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN-Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule-associated protein light chain 3-EGFP to autophagosomes. In N2a cells, OPTN-induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN-Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation-induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a-induced autophagy. These results show that the OPTN-Rab1a complex modulates autophagosome formation in neuroblastoma cells.
视神经病变诱导蛋白（Optineurin，OPTN）是一种自噬受体蛋白，与青光眼和肌萎缩性侧索硬化症有关。OPTN 介导的自噬是一个涉及许多自噬调节蛋白的复杂过程。自噬在清除受损细胞器、细胞内病原体和蛋白质聚集体以维持细胞内平衡方面起着关键作用。我们通过进行大规模酵母-人双杂交测定，鉴定出 Ypt1 是 OPTN 的一个新的相互作用伙伴。免疫共沉淀实验表明，在 N2a 小鼠神经母细胞瘤细胞系中，OPTN 与 Rab1（酵母 Ypt1 的哺乳动物同源物）相互作用。我们通过共聚焦显微镜显示两种蛋白质的细胞内共定位，证实了这种相互作用。我们观察到 OPTN 的锌指结构域对于 Rab1a 结合是重要的。Rab1a 的活性对于与 OPTN 的结合也是必需的。通过测量微管相关蛋白轻链 3-EGFP 向自噬体的易位，确定了 OPTN-Rab1a 复合物在神经元自噬中的作用。在 N2a 细胞中，Rab1a 敲低抑制了 OPTN 诱导的自噬体形成，表明 OPTN-Rab1a 相互作用在神经元自噬过程中起着重要作用。同样，在过表达 Rab1a 的 N2a 细胞中，血清饥饿诱导的自噬体形成增强，而 OPTN 敲低降低了 Rab1a 诱导的自噬。这些结果表明，OPTN-Rab1a 复合物调节神经母细胞瘤细胞中自噬体的形成。
