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.

摘要 血红素蛋白发挥重要的运输，酶和信号作用，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运输成蛋白质 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的传递 在哺乳动物中发生和调节。已经假定了胞质血红素递送蛋白，但是它们的 身份不明。我们发现GAPDH结合睾丸产生的血红素， 是传递到八种不同血红素蛋白靶点所必需的。我们假设依赖于GAPDH的血红素 传递是血红素蛋白功能的基础，我们建议辨别机制，范围， 调节GAPDH依赖的血红素递送。我们的实验利用纯化的蛋白质和细胞培养， 这为分子水平的发现提供了一条便捷的途径，也为验证其生物学特性提供了一种强有力的手段。 本案无关我们有方法控制细胞血红素的产生，监测GAPDH-血红素的结合和转移， 细胞和蛋白质之间，评估血红素向靶标的递送，表征GAPDH-靶标复合物， 确定它们与血红素递送的相关性。 AIM 1. GAPDH如何参与细胞内血红素的传递？我们假设GAPDH可能与 直接与靶蛋白质结合以递送血红素，并发现它与至少四种蛋白质（apo-sGCβ，IDO 1， TDO和Mb）。我们将：（i）确定每个GAPDH-血红素蛋白的界面区域 复合物使用HDx-MS，MS-可裂解的交联，和NMR方法;（ii）使用诱变来测试的作用 GAPDH-靶蛋白接触的血红素交付;（iii）结构特征的GAPDH-血红素复合物 和GAPDH-血红素蛋白复合物;（iv）利用GAPDH-biolD 2-HA 在血红素合成过程中与GAPDH结合的生物素化中间体或中间人蛋白的融合蛋白 （v）部署GAPDH表面电荷突变体以独立地探测GAPDH-靶标结合和在 血红素转移;（vi）用纯化GAPDH和靶蛋白进行血红素转移实验;（vii）筛选 依赖于GAPDH的血红素递送至细胞血红素输出者（FLVCR 1a和ABCG 2）、细胞色素P450（CYP 2D 6 和CYP 3A 4）、两种过氧化物酶（LPO和EPO）、NADPH氧化酶（NOX 5）和血红素加氧酶1和2。 AIM 2.什么控制GAPDH血红素的获取和交付？细胞和分子机制，控制 GAPDH血红素的获得和释放是未知的。我们将：（i）确定线粒体血红素输出蛋白 FLVCR 1b和/或ER血红素输出体PGRMC 2是GAPDH的血红素来源;（ii）测试GAPDH血红素获取是否 涉及与输出者的直接相互作用;（iii）研究细胞中的GAPDH-血红素水平是否受细胞因子的调节。 血红素输出体FLVCR 1a和ABCG 2;（iv）检查是否存在翻译后GAPDH修饰，伴侣蛋白hsp 90， 或唑类药物控制GAPDH血红素的获取和递送。