PDF(Pubmed)
Abstract：
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
摘要：
核因子κB（NF-κB）在多种疾病中起作用。许多炎症信号，如循环脂多糖(LPS)，通过特异性受体激活NF-κB。使用表达NF-κB驱动的自杀基因的LPS处理细胞的全基因组CRISPR-Cas9筛选，我们发现LPS受体Toll样受体4（TLR4）特异性依赖于寡糖转移酶复合物OST-A进行N-糖基化和细胞表面定位。工具化合物NGI-1在体内抑制OST复合物，但是潜在的分子机制仍然未知。我们对STT3A的NGI-1抗性变体进行了CRISPR基础编辑器筛选，OST-A的催化亚基这些变种,结合冷冻电子显微镜研究，揭示NGI-1结合STT3A的催化位点，它捕获供体底物dolichyl-PP-GlcNAc2-Man9-Glc3的分子，表明非竞争性抑制机制。我们的结果为开发STT3A特异性抑制剂提供了理论基础和第一步，并说明了同时进行的碱基编辑器和结构研究定义药物作用机制的能力。
