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 囊蛋白起着重要的运输，酶促和信号传导作用，从根本上影响我们的心血管， 细胞必须将线粒体血红素传输到蛋白质 线粒体之外的成熟和功能。我们的目标是了解细胞内血红素的递送 发生并受到哺乳动物的调节。胞质血红素递送蛋白已发布，但他们的 身份尚不清楚。我们发现GAPDH结合了线粒体生成的血红素，其血红素结合 对于送到八种不同的止血蛋白靶标的至关重要。我们假设GAPDH依赖性血红素 递送是易血蛋白功能至关重要的，我们建议辨别机制，范围和 调节GAPDH依赖性血红素递送。我们的实验利用纯化的蛋白质和细胞培养， 这为分子级发现提供了简便的路径，并且可以验证其生物学的强大手段 关联。我们有控制细胞血红素产生的方法 细胞和蛋白质之间，评估血红素到靶标的血红素递送，表征GAPDH靶络合物，并 确定它们与血红素交付的相关性。 目标1。GAPDH如何参与细胞内血红素递送？我们假设GAPDH可能会结合 直接到靶蛋白输送血红素，并发现至少四个蛋白质（apo-sgcβ，ido1， TDO和MB）在细胞和纯化的形式中。我们将：（i）识别每个GAPDH-蛋白质中的接口区域 使用HDX-MS，MS可裂解的交联和NMR方法的复合物； （ii）使用诱变测试角色 血红素输送中的GAPDH靶蛋白接触； （iii）在结构上表征了GAPDH ​​- 血红素复合物 通过晶体学和单个颗粒EM进行的GAPDH-HEMEPROTEIN复合物； （iv）使用GAPDH-biold2-ha 在血红素期间与GAPDH相关的融合蛋白与生物素化中间或中间人蛋白 转移； （v）将GAPDH表面电荷突变体部署到独立探测GAPDH靶标结合和在 血红素转移； （vi）用纯化的GAPDH和靶蛋白执行血红素转移实验； （vii）屏幕 GAPDH依赖性血红素输送到细胞血红素出口商（FLVCR1A和ABCG2），细胞色素P450（CYP2D6） ＆CYP3A4），两个过氧化物酶（LPO和EPO），一个NADPH氧化酶（NOX5）和血红素氧酶1和2。 目标2。是什么控制了GAPDH血红素的收购和交付？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）确定线粒体血红素出口商是否 FLVCR1b和/或ER血红素出口商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）调查细胞中的GAPDH ​​- 血液水平是否受细胞调节 血红素出口商flvcr1a＆abcg2; （iv）检查如果翻译后的GAPDH修饰，伴侣HSP90， 或Azole药物控制GAPDH血红素的获取和输送。