Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics
Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics
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DOI：
10.1016/j.celrep.2019.04.015
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发表时间：
2019-04-30
期刊：
影响因子：
8.8
通讯作者：
Bennett, Eric J.
中科院分区：
文献类型：
--
作者：
Markmiller, Sebastian;Fulzele, Amit;Bennett, Eric J.
应激颗粒（SG）的形成通常伴随着泛素蛋白酶体系统（UPS）的损伤和泛素化蛋白的积累。SGs，泛素和UPS组件共定位，但泛素途径和SGs之间的关系还没有系统的特点。我们利用泛素或NEDD8激活酶（UAE或NAE）的药理学抑制来探测活性泛素化或neddylation是否调节SG动力学。我们发现，UAE抑制的结果在全球蛋白质泛素化的快速损失，使用泛素特异性蛋白质组学。重要的是，抑制UAE和NAE都不会显著影响SG的形成或分解，表明活性蛋白泛素化或neddylation对SG动力学是不利的。使用抗体与不同的偏好自由泛素或多聚泛素和荧光标记的泛素变体与UAE抑制相结合，我们表明，SGs共定位主要与未缀合的泛素，而不是多聚泛素化蛋白。这些发现阐明了泛素在SG生物学中的作用，并表明游离泛素可能会改变SG蛋白的相互作用。
Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. We utilize pharmacological inhibition of either the ubiquitin- or NEDD8-activating enzyme (UAE or NAE) to probe whether active ubiquitylation or neddylation modulate SG dynamics. We show that UAE inhibition results in rapid loss of global protein ubiquitylation using ubiquitin-specific proteomics. Critically, inhibiting neither UAE nor NAE significantly affected SG formation or disassembly, indicating that active protein ubiquitylation or neddylation is dispensable for SG dynamics. Using antibodies with varying preference for free ubiquitin or polyubiquitin and fluorescently tagged ubiquitin variants in combination with UAE inhibition, we show that SGs co-localize primarily with unconjugated ubiquitin rather than polyubiquitylated proteins. These findings clarify the role of ubiquitin in SG biology and suggest that free ubiquitin may alter SG protein interactions.