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

血红素（Fe protoporphyrin-IX）具有越来越多的重要和动态作用，从催化和电子转移到基因调节和气体传感。然而，血红素是疏水性和细胞毒性的，这意味着它必须在细胞中受到严格的调节控制。短暂的、低亲和力的血红素结合不仅是血红素调节的细胞通路可逆激活/失活的关键，也是监督其毒性反应的关键。因此，对血红素可交换供应的需求已变得明显。我们的目标是阐明血红素供应的机制细节，通过应用一套表征良好的技术，包括荧光寿命成像显微镜（FLIM）、免疫组织化学、靶向siRNA序列的蛋白敲低和蛋白质组学。在这项工作中，我们设计了新的遗传编码血红素传感器，并探索它们在哺乳动物细胞中的部署，以了解血红素的生物利用度。这些传感器由血红素结合域（血红素蛋白，如抗坏血酸过氧化物酶）组成，用荧光标记（如mEGFP或mKATE2）重组表达。与传感器结合的血红素通过福斯特共振能量转移（FRET）淬灭荧光标签，使用荧光寿命成像显微镜（FLIM）测量荧光能量转移，以量化细胞中的生物可利用血红素。我们最近的工作旨在通过培养表达血红素传感器的HEK293细胞，在不同水平的蛋白质表达下探测可交换血红素的供应。特别感兴趣的蛋白质包括甘油醛-3-磷酸脱氢酶（GAPDH）和血红素加氧酶2 (HO-2)，因为它们最近被发现在血红素运输中具有重要意义。也有动机发现额外的血红素伴侣，例如通过蛋白质组学方法。这项工作探讨了这些蛋白质之间的相互作用，以及这些蛋白质的不同表达对血红素生物利用度和细胞功能的影响。我们希望阐明这些基本途径，进一步了解由细胞血红素稳态失衡引起的疾病。该项目由布里斯托尔大学化学学院的Emma Raven教授监督。这项工作是与莱斯特大学安德鲁·哈德森教授及其团队正在进行的合作的一部分。所有的生物成像实验将在布里斯托尔大学的沃尔夫森生物老化中心进行。该项目属于EPSRC化学生物学和生物化学研究领域。

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.