Tri-Institutional MD/PhD Program, Memorial Sloan-Kettering Cancer Center, Weill Cornell Medicine, Rockefeller University, New York, NY 10021, USA.
Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY 10021, USA.
Int J Mol Sci. 2021 Feb 12;22(4):1846. doi: 10.3390/ijms22041846.
Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.
Notch 信号通路对于调控后生动物发育和体内平衡过程中的多种细胞命运决定至关重要。这个独特而高度保守的信号通路依赖于细胞间的接触，从而触发膜锚定转录因子 Notch 的细胞质结构域从膜上的蛋白水解释放。解整合素和金属蛋白酶 (ADAM) 蛋白对于 Notch 的激活至关重要，因为它们可以对其 S2 位点进行加工。虽然 ADAM10 在生理条件下、配体依赖性条件下切割 Notch1，但 ADAM17 主要在无配体条件下切割 Notch1。然而，调节这些 ADAM 在 Notch 加工中独特贡献的机制尚不清楚。我们使用缺乏 ADAM10 和/或 ADAM17 的小鼠胚胎成纤维细胞 (mEF) 的基于细胞的测定，旨在阐明决定 ADAM10 和 ADAM17 对配体依赖性或非依赖性 Notch 加工相对贡献的因素。我们发现，EDTA 刺激的 ADAM17 依赖性 Notch1 加工快速且需要 ADAM17 调节剂 iRhom1 和 iRhom2，而 Delta-like 4 诱导的配体依赖性 Notch1 加工较慢且需要 ADAM10。ADAM17 对 EDTA 诱导的 Notch1 加工的选择性很可能可以通过 ADAM17 对 Notch1 切割位点的偏好以及 EDTA 对 ADAM10 的更强抑制来解释。在 mEF 中，生理上依赖 ADAM10 的 Notch1 加工不能被 ADAM17 代偿，也不能被其他在此处显示能够切割 Notch1 切割位点的 ADAM 代偿，例如 ADAMs9、12 和 19。总的来说，这些结果为 ADAM10 和 ADAM17 对 Notch1 的底物选择性的机制提供了新的见解。
