Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, California, USA.
Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, California, USA; Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, California, USA.
J Biol Chem. 2023 Mar;299(3):102888. doi: 10.1016/j.jbc.2023.102888. Epub 2023 Jan 10.
In several neurodegenerative disorders, the neuronal proteins tau and α-synuclein adopt aggregation-prone conformations capable of replicating within and between cells. To better understand how these conformational changes drive neuropathology, we compared the interactomes of tau and α-synuclein in the presence or the absence of recombinant fibril seeds. Human embryonic stem cells with an inducible neurogenin-2 transgene were differentiated into glutamatergic neurons expressing (1) WT 0N4R tau, (2) mutant (P301L) 0N4R tau, (3) WT α-synuclein, or (4) mutant (A53T) α-synuclein, each genetically fused to a promiscuous biotin ligase (BioID2). Neurons expressing unfused BioID2 served as controls. After treatment with fibrils or PBS, interacting proteins were labeled with biotin in situ and quantified using mass spectrometry via tandem mass tag labeling. By comparing interactions in mutant versus WT neurons and in fibril- versus PBS-treated neurons, we observed changes in protein interactions that are likely relevant to disease progression. We identified 45 shared interactors, suggesting that tau and α-synuclein function within some of the same pathways. Potential loci of shared interactions include microtubules, Wnt signaling complexes, and RNA granules. Following fibril treatment, physiological interactions decreased, whereas other interactions, including those between tau and 14-3-3 η, increased. We confirmed that 14-3-3 proteins, which are known to colocalize with protein aggregates during neurodegeneration, can promote or inhibit tau aggregation in vitro depending on the specific combination of 14-3-3 isoform and tau sequence.
在几种神经退行性疾病中，神经元蛋白 tau 和 α-突触核蛋白会采用易于聚集的构象，从而在细胞内和细胞间进行复制。为了更好地理解这些构象变化如何驱动神经病理学，我们比较了存在或不存在重组纤维种子的情况下 tau 和 α-突触核蛋白的互作组。具有可诱导神经基因 2 转基因的人胚胎干细胞分化为表达（1）WT 0N4R tau、（2）突变（P301L）0N4R tau、（3）WT α-突触核蛋白或（4）突变（A53T）α-突触核蛋白的谷氨酸能神经元，每个基因都与一种混杂的生物素连接酶（BioID2）融合。表达未融合的 BioID2 的神经元作为对照。在用纤维或 PBS 处理后，用生物素原位标记相互作用的蛋白质，并通过串联质量标签标记使用质谱进行定量。通过比较突变体与 WT 神经元以及纤维与 PBS 处理的神经元之间的相互作用，我们观察到与疾病进展相关的蛋白质相互作用的变化。我们确定了 45 个共享的相互作用体，表明 tau 和 α-突触核蛋白在某些相同的途径中发挥作用。潜在的共享相互作用部位包括微管、Wnt 信号复合物和 RNA 颗粒。在纤维处理后，生理相互作用减少，而其他相互作用，包括 tau 和 14-3-3 η 之间的相互作用增加。我们证实，已知在神经退行性变过程中与蛋白聚集体共定位的 14-3-3 蛋白可以根据特定的 14-3-3 同工型和 tau 序列组合促进或抑制 tau 聚集。
