PDF(Pubmed)
Abstract：
Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.
摘要：
支架蛋白有助于介导蛋白质伴侣之间的相互作用，经常优化细胞内信号。在这里，我们使用比较，生物化学，生物物理，分子，和细胞方法来研究支架蛋白NEMO如何促进NF-κB途径的信号传导。来自各种进化上遥远的生物的NEMO和相关蛋白视神经磷酸酶的比较表明，NEMO的中心区域，称为介入域(IVD)，在NEMO和视神经磷酸酶之间保守。先前的研究表明，IVD的中心核心区域是细胞因子诱导的IκB激酶（IKK）活化所必需的。我们表明，视神经磷酸酶的类似区域可以在功能上替代NEMOIVD的核心区域。我们还表明，完整的IVD是形成二硫键结合的NEMO二聚体所必需的。此外，该核心区的失活突变消除了NEMO在体外形成泛素诱导的液-液相分离液滴和在体内形成信号诱导的斑点的能力。截短的NEMO变体的热和化学变性研究表明，IVD，虽然本质上不会破坏稳定，会降低NEMO周围区域的稳定性，由于通过侧翼上游和下游域赋予该区域的结构需求冲突。IVD中的这种构象应变介导NEMO的N端和C端区域之间的变构通信。总的来说,这些结果支持了NEMO的IVD通过充当NEMO构象变化的介质参与信号诱导的IKK/NF-κB通路激活的模型.
