This work demonstrates the reconstitution of active methylxanthine synthesis enzymes in human cells and their potential use as inducible reporter enzymes. A variety of plant enzymes involved in caffeine synthesis have been characterized in vitro and several of these methylxanthine synthesis enzymes have been heterologously-expressed in yeast or bacteria. In this work, enzymes from Coffea arabica, Camellia sinensis, and Paullinia cupana have been heterologously-expressed in human cells. We demonstrate that the enzymes tested exhibit similar patterns of activity with a set of xanthine substrates in human cells when compared to previous reports of in vitro activity. We demonstrate that the activity of these enzymes can be used as a reporter for juxtacrine signaling using synNotch-induced expression in the presence of an appropriate substrate. When used in combination with synthetic caffeine receptors, this work has potential for use as an in vivo reporter (e.g. enabling non-invasive monitoring of cell-cell interactions after a cellular transplant) or in synthetic intercellular signaling a methylxanthine, such as caffeine, acting as a synthetic paracrine hormone.

中文翻译：

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甲基黄嘌呤合成酶在哺乳动物细胞中的异源表达及其作为报告蛋白的用途

这项工作证明了人类细胞中活性甲基黄嘌呤合成酶的重构及其作为诱导型报告酶的潜在用途。已经在体外表征了涉及咖啡因合成的多种植物酶，并且这些甲基黄嘌呤合成酶中的几种已经在酵母或细菌中异源表达。在这项工作中，来自阿拉伯咖啡，山茶和泡桐的酶  已经在人类细胞中异源表达。我们证明与以前的体外活性报告相比，所测试的酶在人细胞中显示出与一组黄嘌呤底物相似的活性模式。我们证明了这些酶的活性可以用作在适当底物存在下使用synNotch诱导表达的邻苯并林信号转导的报告基因。当与合成咖啡因受体结合使用时，这项工作有可能用作体内报道分子（例如，在细胞移植后能够无创监测细胞间相互作用）或在合成细胞间信号传导甲基黄嘌呤（如咖啡因）作为合成的旁分泌激素。