Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

中文翻译：

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NRARP对Notch细胞内结构域锚蛋白重复序列​​堆栈的扩展促进了Notch信号的反馈抑制。

规范的Notch信号转导依赖于Notch受体的蛋白水解作用，以产生诱导Notch反应性基因转录的核效应子。在高等生物中，在许多不同细胞类型中激活的一种Notch反应基因编码Notch调节的锚蛋白重复蛋白（NRARP），它充当Notch反应的负反馈调节剂。在这里，我们表明NRARP抑制Notch依赖性T细胞急性淋巴细胞白血病（T-ALL）细胞系的生长，并直接与核心Notch转录激活复合物（NTC）结合，需要转录因子RBPJ和Notch细胞内结构域（NICD），而不是像Mastermind一样的蛋白质或DNA。NRARP-NICD1-RBPJ-DNA复合物的晶体结构，已确定为3.75Å分辨率，揭示了NRARP-NICD1-RBPJ复合物的组装依赖于RBPJ和NICD1的同时结合，NRARP的三个锚蛋白重复序列​​扩展了Notch1锚蛋白重复序列​​栈。NRARP-NICD1接口处的突变破坏了蛋白质进入NTC的进入，并在培养细胞的Notch信号测定中废除了反馈抑制作用。NRARP的强迫表达降低了细胞中NICD的含量，这表明NRARP可能促进了NICD的降解。这些研究建立了NRARP参与NTC的结构基础，并为调节Notch信号的关键负反馈机制提供了见识。NRARP-NICD1接口处的突变破坏了蛋白质进入NTC的进入，并在培养细胞的Notch信号测定中废除了反馈抑制作用。NRARP的强迫表达降低了细胞中NICD的含量，这表明NRARP可能促进了NICD的降解。这些研究建立了NRARP参与NTC的结构基础，并为调节Notch信号的关键负反馈机制提供了见识。NRARP-NICD1接口处的突变破坏了蛋白质进入NTC的进入，并在培养细胞的Notch信号测定中废除了反馈抑制作用。NRARP的强迫表达降低了细胞中NICD的含量，这表明NRARP可能促进了NICD的降解。这些研究建立了NRARP参与NTC的结构基础，并为调节Notch信号的关键负反馈机制提供了见识。