美国拉霍亚免疫学研究所Samuel A. Myers研究小组发现，以翻译后修饰为中心的碱基编辑器筛选可高通量评估磷酸化位点功能。相关论文于2024年4月29日在线发表在《自然—方法学》杂志上。

研究人员介绍了通过开发以翻译后修饰（PTM）为中心的碱基编辑技术，并结合表型筛选，在时间分辨磷酸化蛋白质组学的指导下，对磷酸化位点进行高通量的功能评估。以T细胞活化为模型，研究人员观察到了数百个未研究的磷酸化位点，这些位点调节着NFAT的转录活性。研究人员发现了磷酸化介导的PHLPP1核定位，它能促进NFAT但抑制NFκB的活性。

研究人员还发现，特定的磷酸化突变体能以微妙而独特的模式改变基因表达，显示了微调转录反应的潜力。总之，PTM位点的碱基编辑器筛选为剖析信号通路中的PTM功能提供了一个强大的平台。

据悉，驱动基因表达的信号通路通常被描述为具有十几个标志性磷酸化和转录事件。实际上，数以千计的动态PTM几乎协调着每一种细胞功能，而人们缺乏技术来发现这些庞大的生化通路与遗传回路之间的因果联系。

附：英文原文

Title: Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput

Author: Kennedy, Patrick H., Alborzian Deh Sheikh, Amin, Balakar, Matthew, Jones, Alexander C., Olive, Meagan E., Hegde, Mudra, Matias, Maria I., Pirete, Natan, Burt, Rajan, Levy, Jonathan, Little, Tamia, Hogan, Patrick G., Liu, David R., Doench, John G., Newton, Alexandra C., Gottschalk, Rachel A., de Boer, Carl G., Alarcn, Suzie, Newby, Gregory A., Myers, Samuel A.

Issue&Volume: 2024-04-29

Abstract: Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using Tcell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

DOI: 10.1038/s41592-024-02256-z

Source: https://www.nature.com/articles/s41592-024-02256-z