Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2’s catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2’s gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (kcat/KM = 830 ± 40 M-1 s-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (kcat/KM = 90 ± 9 M-1 s-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1% O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

中文翻译：

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脯氨酰羟化酶的气道工程重新编程细胞中的缺氧信号


细胞已经进化出复杂的机制来识别和响应氧气 (O2) 浓度的变化。在这里，我们通过脯氨酰羟化酶 2 (PHD2)（一种非血红素铁依赖性 O2 传感器）的气道工程重新编程了细胞缺氧（低 O2）信号。利用计算模型和蛋白质工程技术，我们确定了气体通道和其中限制 O2 流向 PHD2 催化核心的关键残留物。我们表明，对这些残基进行系统修饰可以打开 PHD2 气体通道的收缩拓扑。使用 NO 作为替代双原子气体的动力学停流测量，我们证明其与隧道工程突变体的铁中心的关联率提高了 3.5 倍。与 WT PHD2 (kcat/KM = 90 ± 9 M-1 s-1)。此外，在 HEK-293T 哺乳动物细胞中表达设计的 PHD2 突变体的质粒转染表明，在 1% O2 的缺氧条件下，HIF-1α 和下游缺氧反应转录本显着减少。总体而言，这些研究强调 PHD2 的激活是重新编程细胞中缺氧反应和 HIF 信号传导的新途径。