Defining a pathway for mitochondrial heme trafficking

定义线粒体血红素运输途径

• 批准号: 10733705
• 负责人: DENNIS J STUEHR
• 金额: $ 54.42万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10733705
• 关键词: ABCG2 gene; Anabolism; Anemia; Asthma; Autoimmunity; Azoles; Binding; Biological; Biological Process; Biotinylation; CYP2D6 gene; CYP3A4 gene; Cardiovascular system; Cell Culture Techniques; Cell membrane; Cells; Charge; Chimeric Proteins; Complex; Crystallization; Crystallography; Cytochrome P450; Dioxygenases; Disease; Drug Controls; Electron Transport; Enzymes; Eukaryota; Event; Goals; Health; Heme; Hemeproteins; Hemoglobin; Hereditary Disease; Human; Immune system; Infection; Iron; Kinetics; Knowledge; Lung; Mammalian Cell; Mammals; Medical; Mission; Mitochondria; Modification; Molecular; Molecular Chaperones; Monitor; Mutagenesis; Myoglobin; NADPH Oxidase; National Institute of General Medical Sciences; Neurologic; Nitric Oxide Synthase; Organism; Pathway interactions; Peroxidases; Pharmaceutical Preparations; Physiology; Play; Production; Property; Proteins; Publishing; Regulation; Research; Role; Signal Transduction; Site; Soluble Guanylate Cyclase; Source; Steroid biosynthesis; Structure; Surface; System; Testing; Transport Process; Vasodilation; Work; Yeasts; cofactor; crosslink; experimental study; heme a; heme oxygenase-1; heme oxygenase-2; indoleamine; mutant; particle; protein complex; protein function; protein purification; reconstitution; sensor; trafficking; tumor

ABSTRACT Hemeproteins play vital transport, enzymatic, and signaling roles that fundamentally impact our cardiovascular, pulmonary, digestive, neurological, and immune systems. Cells must transport mitochondrial heme to proteins that mature and function outside the mitochondria. Our goal is to understand how intracellular heme delivery takes place and is regulated in mammals. Cytosolic heme delivery proteins have been postulated but their identities are unclear. We found that GAPDH binds mitochondrially-generated heme and that its heme binding is essential for delivery to eight different hemeprotein targets. We hypothesize that GAPDH-dependent heme delivery is fundamental for hemeprotein function, and we propose to discern mechanisms, scope, and regulation of GAPDH-dependent heme delivery. Our experiments utilize purified proteins and cell culture, which provides a facile path to molecular-level discoveries and a robust means to validate their biological relevance. We have ways to control cell heme production, monitor GAPDH-heme binding and transfer in live cells and between proteins, assess heme delivery to targets, characterize GAPDH-target complexes, and determine their relevance for heme delivery. AIM 1. How does GAPDH participate in intracellular heme delivery? We hypothesize GAPDH may bind directly to the target proteins to deliver heme and found it does so with at least four proteins (apo-sGCβ, IDO1, TDO, and Mb) in cells and purified form. We will: (i) identify interface regions in each GAPDH-hemeprotein complex using HDx-MS, MS-cleavable crosslinking, and NMR approaches; (ii) use mutagenesis to test the role of GAPDH-target protein contacts in heme deliveries; (iii) structurally characterize the GAPDH-heme complex and GAPDH-hemeprotein complexes by crystallography and single particle EM; (iv) utilize a GAPDH-biolD2-HA fusion protein to biotinylate intermediate or middleman proteins that associate with GAPDH during heme transfers; (v) deploy GAPDH surface charge mutants to independently probe GAPDH-target binding and role in heme transfers; (vi) perform heme transfer experiments with purified GAPDH & target proteins; (vii) screen for GAPDH-dependent heme deliveries to cell heme exporters (FLVCR1a & ABCG2), cytochrome P450’s (CYP2D6 & CYP3A4), two peroxidases (LPO & EPO), an NADPH oxidase (NOX5), and heme oxygenases 1 & 2. AIM 2. What controls GAPDH heme acquisition & deliveries? Cell & molecular mechanisms that control GAPDH heme acquisition and release are unknown. We will: (i) determine if mitochondrial heme exporter FLVCR1b and/or ER heme exporter PGRMC2 is a heme source for GAPDH; (ii) test if GAPDH heme acquisition involves direct interaction with the exporters; (iii) investigate if GAPDH-heme level in cells is regulated by cell heme exporters FLVCR1a & ABCG2; (iv) examine if post-translational GAPDH modification, chaperone hsp90, or azole drugs control GAPDH heme acquisition and delivery.

抽象的 血红素蛋白发挥重要的运输、酶促和信号传导作用，从根本上影响我们的心血管、 肺、消化、神经和免疫系统。细胞必须将线粒体血红素转运为蛋白质 在线粒体外成熟并发挥作用。我们的目标是了解细胞内血红素传递如何 在哺乳动物中发生并受到调节。胞质血红素递送蛋白已被假设，但它们 身份不明。我们发现 GAPDH 结合线粒体生成的血红素，并且它的血红素结合 对于递送八种不同的血红素蛋白靶标至关重要。我们假设 GAPDH 依赖性血红素 递送是血红素蛋白功能的基础，我们建议辨别机制、范围和 调节 GAPDH 依赖性血红素输送。我们的实验利用纯化的蛋白质和细胞培养物， 这为分子水平的发现提供了一条简便的途径，并为验证其生物学特性提供了强有力的手段 关联。我们有方法控制细胞血红素的产生、监测 GAPDH-血红素的结合和活体转移 细胞和蛋白质之间，评估血红素向靶标的传递，表征 GAPDH-靶标复合物，以及 确定它们与血红素传递的相关性。 目的 1. GAPDH 如何参与细胞内血红素递送？我们假设 GAPDH 可能结合 直接向目标蛋白质递送血红素，并发现它至少与四种蛋白质（apo-sGCβ、IDO1、 TDO 和 Mb）以细胞形式和纯化形式存在。我们将： (i) 识别每个 GAPDH-血红素蛋白的界面区域 使用 HDx-MS、MS 可裂解交联和 NMR 方法形成复合物； (ii) 使用诱变来测试作用 血红素输送中 GAPDH 与靶蛋白的接触； (iii) 表征 GAPDH-血红素复合物的结构 通过晶体学和单粒子 EM 分析 GAPDH-血红素蛋白复合物； (iv) 利用 GAPDH-biolD2-HA 融合蛋白对血红素过程中与 GAPDH 相关的中间体或中间蛋白进行生物素化 转账； (v) 部署 GAPDH 表面电荷突变体以独立探测 GAPDH 靶标结合及其作用 血红素转移； (vi) 用纯化的 GAPDH 和靶蛋白进行血红素转移实验； (vii) 筛选 GAPDH 依赖性血红素向细胞血红素输出蛋白 (FLVCR1a 和 ABCG2)、细胞色素 P450 (CYP2D6) 的输送 和 CYP3A4)、两种过氧化物酶（LPO 和 EPO）、一种 NADPH 氧化酶 (NOX5) 以及血红素加氧酶 1 和 2。 目标 2. 什么控制 GAPDH 血红素的获取和传递？控制的细胞和分子机制 GAPDH 血红素的获取和释放尚不清楚。我们将：（i）确定线粒体血红素输出者是否 FLVCR1b 和/或 ER 血红素输出蛋白 PGRMC2 是 GAPDH 的血红素来源； (ii) 测试 GAPDH 血红素是否获得 涉及与出口商的直接互动； (iii) 研究细胞中的GAPDH-血红素水平是否受细胞调节 血红素出口商 FLVCR1a 和 ABCG2； (iv) 检查翻译后 GAPDH 修饰、分子伴侣 hsp90、 或唑类药物控制 GAPDH 血红素的获取和传递。