Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536.
Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40536.
Proc Natl Acad Sci U S A. 2018 Dec 18;115(51):E11904-E11913. doi: 10.1073/pnas.1810413115. Epub 2018 Nov 19.
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by preferential motor neuron death. Approximately 15% of ALS cases are familial, and mutations in the () gene contribute to a subset of familial ALS cases. FUS is a multifunctional protein participating in many RNA metabolism pathways. ALS-linked mutations cause a liquid-liquid phase separation of FUS protein in vitro, inducing the formation of cytoplasmic granules and inclusions. However, it remains elusive what other proteins are sequestered into the inclusions and how such a process leads to neuronal dysfunction and degeneration. In this study, we developed a protocol to isolate the dynamic mutant FUS-positive cytoplasmic granules. Proteomic identification of the protein composition and subsequent pathway analysis led us to hypothesize that mutant FUS can interfere with protein translation. We demonstrated that the ALS mutations in FUS indeed suppressed protein translation in N2a cells expressing mutant FUS and fibroblast cells derived from FUS ALS cases. In addition, the nonsense-mediated decay (NMD) pathway, which is closely related to protein translation, was altered by mutant FUS. Specifically, NMD-promoting factors UPF1 and UPF3b increased, whereas a negative NMD regulator, UPF3a, decreased, leading to the disruption of NMD autoregulation and the hyperactivation of NMD. Alterations in NMD factors and elevated activity were also observed in the fibroblast cells of FUS ALS cases. We conclude that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation, both of which likely contribute to motor neuron death.
肌萎缩侧索硬化症（ALS）是一种不可治愈的神经退行性疾病，其特征是运动神经元优先死亡。大约 15%的 ALS 病例是家族性的，并且 () 基因的突变导致一部分家族性 ALS 病例。FUS 是一种多功能蛋白，参与许多 RNA 代谢途径。ALS 相关突变导致 FUS 蛋白在体外发生液-液相分离，诱导细胞质颗粒和包涵体的形成。然而，目前仍不清楚还有哪些蛋白质被包含在包涵体中，以及这种过程如何导致神经元功能障碍和退化。在这项研究中，我们开发了一种分离动态突变 FUS 阳性细胞质颗粒的方案。对蛋白质组成的蛋白质组学鉴定和随后的途径分析使我们假设突变 FUS 可以干扰蛋白质翻译。我们证明，FUS 中的 ALS 突变确实抑制了表达突变 FUS 的 N2a 细胞和源自 FUS ALS 病例的成纤维细胞中的蛋白质翻译。此外，与蛋白质翻译密切相关的无义介导的衰变（NMD）途径也被突变 FUS 改变。具体而言，NMD 促进因子 UPF1 和 UPF3b 增加，而负 NMD 调节剂 UPF3a 减少，导致 NMD 自身调节的破坏和 NMD 的过度激活。在 FUS ALS 病例的成纤维细胞中也观察到 NMD 因子的改变和活性升高。我们得出结论，突变 FUS 抑制蛋白质生物合成并破坏 NMD 调节，这两者都可能导致运动神经元死亡。
