Role of monoubiquitylation on the control of IκBα degradation and NF-κB activity.
Role of monoubiquitylation on the control of IκBα degradation and NF-κB activity.
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DOI：
10.1371/journal.pone.0025397
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Lang V
中科院分区：
文献类型：
--
作者：
Da Silva-Ferrada E;Torres-Ramos M;Aillet F;Campagna M;Matute C;Rivas C;Rodríguez MS;Lang V
NF-κB 通路受多种翻译后修饰调节，包括磷酸化、泛素化和 SUMO 化。许多这些修饰作用于天然抑制剂 IκBα，调节其控制信号介导的 NF-κB 活性的能力。虽然涉及 IκBα 磷酸化和多泛素化的经典途径已得到很好的表征，但这些翻译后修饰在控制基础 NF-κB 活性中的作用尚未得到深入探讨。使用最近开发的串联重复泛素结合实体（也称为泛素陷阱）来捕获泛素化蛋白质，我们从多种大鼠器官和细胞类型中鉴定了 IκBα 的单泛素化形式。这些形式的鉴定通过不同的程序进行了证明，例如使用特定泛素抗体进行免疫沉淀或 His6-泛素下拉。 IκBα 的单泛素化形式能够抵抗 TNFα 介导的降解，并且即使在蛋白酶体抑制剂处理后也可以使用 TUBE 捕获。与单SUMO化一样，单泛素化不依赖于丝氨酸32/36上的IκBα磷酸化，并且在TNFα刺激后不会最佳降解。泛素-IκBα 融合体表现出磷酸化缺陷和对 TNFα 介导的降解的抵抗力，与内源性单泛素化 IκBα 观察到的情况类似。 IκBα 融合体上单个泛素部分的 N 端连接导致与 IKKβ 激酶的结合缺陷和 SCF 连接酶成分 βTrCP 的募集，从而对 NF-κB 活性产生负面影响。总而言之，我们的结果表明存在对 TNFα 诱导的蛋白水解具有抗性的单泛素化 IκBα 库，它能够相互作用并抑制 DNA 结合和 NF-κB 转录活性。这种 IκBα 库可能在控制基础和信号介导的 NF-κB 活性中发挥重要作用。
The NF-κB pathway is regulated by multiple post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. Many of these modifications act on the natural inhibitor IκBα modulating its capacity to control signal-mediated NF-κB activity. While the canonical pathway involving the phosphorylation and polyubiquitylation of IκBα has been well characterized, the role of these post-translational modifications in the control of basal NF-κB activity has not been deeply explored. Using the recently developed Tandem-repeated Ubiquitin Binding Entities (also known as ubiquitin traps) to capture ubiquitylated proteins, we identified monoubiquitylated forms of IκBα from multiple rat organs and cell types. The identification of these forms was demonstrated through different procedures such as immunoprecipitations with specific ubiquitin antibodies or His6-Ubiquitin pull downs. Monoubiquitylated forms of IκBα are resistant to TNFα-mediated degradation and can be captured using TUBEs, even after proteasome inhibitors treatment. As it occurs for monoSUMOylation, monoubiquitylation is not dependent of the phosphorylation of IκBα on the serines 32/36 and is not optimally degraded after TNFα stimulation. A ubiquitin-IκBα fusion exhibits phosphorylation defects and resistance to TNFα mediated degradation similar to the ones observed for endogenous monoubiquitylated IκBα. The N-terminal attachment of a single ubiquitin moiety on the IκBα fusion results in a deficient binding to the IKKβ kinase and recruitment of the SCF ligase component βTrCP, promoting a negative impact on the NF-κB activity. Altogether, our results suggest the existence of a reservoir of monoubiquitylated IκBα resistant to TNFα-induced proteolysis, which is able to interact and repress DNA binding and NF-κB transcriptional activity. Such pool of IκBα may play an important role in the control of basal and signal-mediated NF-κB activity.