Shandong Provincial Key Laboratory of Animal Resistance Biology, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Institute of Biomedical Sciences, Shandong Normal University, Jinan, 250014, China.
State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials of the Ministry of Education, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, 300071, China.
Sci China Life Sci. 2021 Apr;64(4):575-583. doi: 10.1007/s11427-020-1765-6. Epub 2020 Jul 28.
Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions, and oxidative damage has been implicated in several neurodegenerative diseases. End-binding protein 1 (EB1) is a master regulator of microtubule plus-end tracking proteins (+TIPs) and is critically involved in the control of microtubule dynamics and cellular processes. However, the impact of glutathionylation on EB1 functions remains unknown. Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation. In vitro biochemical and cellular assays reveal that EB1 is glutathionylated. Diamide, a mild oxidizing reagent, reduces EB1 comet number and length in cells, indicating the impairment of microtubule dynamics. Three cysteine residues of EB1 are glutathionylated, with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics. In addition, glutaredoxin 1 (Grx1) deglutathionylates EB1, and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration, suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity. Collectively, these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.
细胞骨架蛋白在氧化条件下容易发生谷胱甘肽化，氧化损伤与几种神经退行性疾病有关。末端结合蛋白 1（EB1）是微管末端追踪蛋白（+TIPs）的主要调节因子，对于控制微管动力学和细胞过程至关重要。然而，谷胱甘肽化对 EB1 功能的影响尚不清楚。在这里，我们揭示了谷胱甘肽化对于控制 EB1 活性和防止 EB1 不可逆氧化是很重要的。体外生化和细胞实验表明 EB1 发生了谷胱甘肽化。二亚胺，一种温和的氧化试剂，减少了细胞中 EB1 彗星的数量和长度，表明微管动力学受损。EB1 的三个半胱氨酸残基发生了谷胱甘肽化，这些半胱氨酸残基的突变削弱了微管动力学，但缓冲了二亚胺诱导的微管动力学下降。此外，谷胱甘肽还原酶 1（Grx1）使 EB1 去谷胱甘肽化，Grx1 耗竭抑制微管动力学，并导致细胞分裂方向和细胞迁移缺陷，表明 Grx1 介导的去谷胱甘肽化在维持 EB1 活性方面起着关键作用。总的来说，这些数据揭示了 EB1 谷胱甘肽化是调节微管动力学和基于微管的细胞活动的重要保护机制。
