Heterologous expression of methylxanthine synthesis enzymes in mammalian cells and their use as reporter proteins

Abstract

AbstractThis 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 characterizedin vitroand several of these methylxanthine synthesis enzymes have been heterologously-expressed in yeast or bacteria. In this work, enzymes fromCoffea arabica, Camellia sinensis, andPaullinia cupanahave 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 ofin vitroactivity. 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 anin vivoreporter (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.