Using cellular reporters to probe the homeostasis of heme in the cell

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Heme (Fe protoporphyrin-IX) has an ever-growing number of essential and dynamic roles, from catalysis and electron transfer to gene modulation and gas sensing. However, heme is hydrophobic and cytotoxic, meaning it must be under tight regulatory control in the cell. Transient, low affinity heme binding is key not only for the reversible activation/inactivation of heme-regulated cellular pathways but also to oversee its toxic reactivity. Thus, the need for of an exchangeable supply of heme has become apparent. We aim to elucidate the mechanistic details of the heme supply by applying a set of well-characterised techniques, including fluorescent lifetime imaging microscopy (FLIM), immunohistochemistry, protein knockdowns with targeted siRNA sequences and proteomics.In this work we design novel genetically encoded heme sensors and explore their deployment in mammalian cells to understand heme bioavailability. These sensors consist of a heme binding domain (a heme protein, such as ascorbate peroxidase) expressed recombinantly with a fluorescent tag (such as mEGFP or mKATE2). Heme binding to the sensor quenches the fluorescent tag through Forster resonance energy transfer (FRET), which is measured using fluorescence lifetime imaging microscopy (FLIM) to quantify bioavailable heme in the cell. Our recent work aims to probe the exchangeable heme supply, by growing HEK293 cells - expressing a heme sensor - under varying levels of protein expression. Proteins of particular interest include glyceraldehyde-3-phoshphate dehydrogenase (GAPDH) and heme oxygenase 2 (HO-2) because of their recently uncovered significance in heme trafficking. There is also motivation to uncover additional heme chaperones, for example through proteomics methods. This work explores the interplay between these proteins, and the consequences that varying expression of these proteins has on heme bioavailability and cell function. We hope to shed light on these fundamental pathways, furthering our understanding of disorders resulting from imbalances in cellular heme homeostasis.The project is under the supervision of Professor Emma Raven in the School of Chemistry, University of Bristol. The work is part of an ongoing collaboration with Professor Andrew Hudson and his group at the University of Leicester. All bioimaging experiments will be undertaken at the Wolfson Biomaging Centre at the University of Bristol. This project falls within the Chemical Biology and Biological Chemistry EPSRC research area.