Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling
Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling
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DOI：
10.1126/scisignal.aay2369
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发表时间：
2019-11-05
影响因子：
7.3
通讯作者：
Blacklow, Stephen C.
中科院分区：
文献类型：
--
作者：
Jarrett, Sanchez M.;Seegar, Tom C. M.;Blacklow, Stephen C.
典型的Notch信号传导依赖于Notch受体的受调节的蛋白水解来产生诱导Notch反应基因转录的核效应子。在高等生物体中，一种在许多不同细胞类型中被激活的 Notch 响应基因编码 Notch 调节的锚蛋白重复蛋白 (NRARP)，该蛋白充当 Notch 响应的负反馈调节因子。在这里，我们发现 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 胞内结构域 (NICD)，但不需要 Mastermind 样蛋白或 DNA。 NRARP-NICD1-RBPJ-DNA 复合物的晶体结构确定为 3.75 埃分辨率，表明 NRARP-NICD1-RBPJ 复合物的组装依赖于 RBPJ 和 NICD1 的同时接合，其中 NRARP 的三个锚蛋白重复序列延伸了 Notch1 锚蛋白重复序列堆栈。 NRARP-NICD1 界面的突变破坏了蛋白质进入 NTC 的过程，并消除了培养细胞中 Notch 信号传导测定中的反馈抑制。 NRARP的强制表达降低了细胞中NICD的丰度，表明NRARP可能促进NICD的降解。这些研究奠定了 NRARP 参与 NTC 的结构基础，并提供了对调节 Notch 信号传导的关键负反馈机制的见解。
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 angstrom 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.