Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1^−/− background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

中文翻译：

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人POFUT1的结构，其对不依赖配体的致癌Notch信号传导的要求以及Dowling-Degos突变的功能作用

蛋白O-岩藻糖基转移酶-1（POFUT1）可将岩藻糖残基转移至受体蛋白表皮生长因子样重复序列的丝氨酸和苏氨酸残基上的受体位点，对哺乳动物的Notch信号转导至关重要。在这里，我们检查了POFUT1缺失对与人Notch1某些与白血病相关的突变相关的致癌信号的后果，报告了人POFUT1在自由和GDP结合状态下的结构，并评估了Dowling-Degos突变对人的影响POFUT1功能。CRISPR介导的POFUT1的敲除在U2OS细胞中，抑制正常的Notch1信号传导和与Notch1的致白血病突变相关的配体非依赖性信号传导都受到抑制。野生型POFUT1可拯救正常和致癌信号，但活性位点R240A突变会损害拯救。人类酶的总体结构与秀丽隐杆线虫蛋白的结构非常相似，尽管成熟蛋白的主要序列同一性仅为39％，但总体骨架RMSD为0.93Å。GDP-岩藻糖与人酶的结合诱导有限的骨架构象运动，尽管R43和D244的侧链重新定向以与复合物中的岩藻糖部分直接接触。除M262T外，已报道的POFUT1的Dowling-Degos突变未能在CRISPR工程中有效挽救Notch1信号转导POFUT1 ^-/-背景。总之，这些研究确定POFUT1是Notch1突变驱动的癌症的潜在靶标，并为其抑制提供了结构路线图。