Maduka Austin O, Manohar Sandhya, Foster Matthew W, Silva Gustavo M
Department of Biology, Duke University, Durham, North Carolina, USA.
Department of Biology, Institute for Biochemistry, ETH Zürich, Zürich, Switzerland.
Mol Cell Proteomics. 2025 Mar;24(3):100920. doi: 10.1016/j.mcpro.2025.100920. Epub 2025 Jan 28.
Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote concerted response mechanisms remain understudied. Here, we show that K63-linked polyubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in noncytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of noncytosolic compartments expanded 2.5-fold the pool of proteins (2,494) and provided a comprehensive number of sites (10,157) known to be ubiquitinated during arsenite stress, suggesting their involvement in a myriad of cellular pathways. Moreover, subcellular proteome analyses revealed proteins that are recruited to noncytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase valosin-containing protein (VCP) that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to noncytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of noncytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.
在应激条件下，细胞会重新编程其分子机制以减轻损伤并促进存活。在氧化应激期间，泛素信号在整体上会增强，它控制着蛋白质的命运，并在几个亚细胞区室中支持应激防御。然而，驱动亚细胞泛素定位以促进协同反应机制的规则仍未得到充分研究。在这里，我们表明，已知能促进不依赖蛋白酶体途径的K63连接的多聚泛素链，在亚砷酸钠诱导的哺乳动物细胞氧化应激期间主要积聚在非胞质区室中。我们对非胞质区室进行的亚细胞泛素蛋白质组分析将蛋白质库（2494种）扩大了2.5倍，并提供了已知在亚砷酸盐应激期间会发生泛素化的全面位点数量（10157个），这表明它们参与了无数细胞途径。此外，亚细胞蛋白质组分析揭示了在应激条件下被招募到非胞质区室的蛋白质，包括富含含缬酪肽蛋白（VCP）的辅助泛素结合衔接子，VCP可处理泛素化底物以进行下游信号传导。我们进一步表明，在亚砷酸盐应激下，VCP通过其衔接子NPLOC4以泛素依赖的方式被招募到非胞质区室。此外，我们表明VCP和NPLOC4的活性对于维持低水平的非胞质K63连接的泛素链至关重要，这支持了一个被活性氧破坏的泛素结合和去除的循环模型。这项工作加深了我们对局部泛素和VCP信号在应激反应基本机制中的作用的理解，并突出了在动态环境下重塑人类亚细胞蛋白质组的组成和功能所必需的新途径和分子参与者。
