New mechanism and regulation of intracellular heme delivery in mammals

哺乳动物细胞内血红素输送的新机制和调控

• 批准号: 8082579
• 负责人: DENNIS J STUEHR
• 金额: $ 29.83万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8082579
• 关键词: Anemia due to Chronic Disorder; Animals; Binding; Biochemical; Biology; Blood; Cell physiology; Cells; Crystallography; Cytochrome P450; Disease; Enzymes; Gases; Glutathione S-Transferase; Glyceraldehyde-3-Phosphate Dehydrogenases; Goals; Health; Heme; Hemeproteins; Hemoglobin; Homeostasis; Human; In Vitro; Knowledge; Mammalian Cell; Mammals; Measures; Medicine; Modeling; Molecular; Nitric Oxide; Nitric Oxide Synthase; Nitrosation; Organ; Pharmaceutical Preparations; Physiology; Play; Point Mutation; Process; Property; Protein S; Proteins; Reaction; Regulation; Role; SKIL gene; Spectrum Analysis; Staging; Structure; System; Testing; Thioredoxin; Touch sensation; catalase; enzyme activity; heme oxygenase-1; human NOS2A protein; mutant; nitric oxide reductase; novel; protein complex; reconstitution; response

DESCRIPTION (provided by applicant): Heme proteins play vital roles in physiology and medicine. Our broad goal is to understand the mechanisms of intracellular heme delivery and insertion into cytosolic proteins, and how this process is regulated in mammalian cells. We found that nitric oxide (NO) blocks cellular heme insertion into a range of heme proteins (NO synthases, cytochrome P450's, hemoglobin, and catalase). Using this information, and inducible NO synthase (iNOS) as our model heme protein, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key player in the process. Our initial studies suggest that NO inhibits cellular heme insertion into iNOS by promoting a specific S-nitrosation of GAPDH, thereby altering GAPDH properties that are independent of its enzyme activity. We hypothesize that GAPDH plays an essential role in intracellular heme delivery through a non-traditional function that is regulated by its S-nitrosation. We propose cellular, biochemical, and biophysical approaches to test three aspects of our general hypothesis: Aim 1. Does GAPDH play a general role in intracellular heme delivery & homeostasis? We will test this by determining if GAPDH is involved in heme insertion into four proteins (hemoglobin, cytochrome P450, constitutive NOS, and catalase), and if GAPDH is involved in heme delivery to heme oxygenase 1 & 2. Aim 2. Do cells control their heme insertion reactions by regulating their capacity for protein S-nitrosation? We will test this by: (i) up or down-regulating the expression level of known cell denitrosylase enzymes (Thioredoxin-1 and GSH-NO reductase) to alter buildup of cellular S-nitrosoproteins (protein-SNO) in response to NO, and then (ii) determining if these changes alter levels of total protein-SNO and SNO-GAPDH in cells, and shift the NO sensitivity of cellular heme insertion in predictable ways. Aim 3. How are the heme binding, heme transfer, and protein interaction properties of GAPDH related to cellular heme delivery? We will investigate this by: (i) Measuring the heme binding parameters of pure GAPDH proteins that differ in their ability to support heme insertion in cells (wild type, S-nitrosated, point mutants), (ii) Evaluating interaction of GAPDH proteins with apo-protein targets and their ability to transfer bound heme in an in-vitro reconstitution system, and (iii) Solving the structure of the GAPDH-heme complex by protein crystallography and spectroscopy. Together, our studies will discern novel roles for NO, protein S-nitrosation, and GAPDH in intracellular heme delivery and insertion into proteins. This will advance our understanding of a fundamental process at the cellular and molecular levels, and will set the stage to investigate how these facets impact human health and disease at the organ and whole animal level. PUBLIC HEALTH RELEVANCE: Heme proteins exist in our cells and have many essential roles in our bodies. Our study will fill a tremendous gap in knowledge regarding how heme proteins are made in our cells, and how this process is regulated by nitric oxide, a gas that is made naturally by our bodies as a control agent. Our findings will touch many aspects of human health and disease, including blood formation, drug response, anemia, and inflammation.

描述（由申请人提供）：血红素蛋白在生理学和医学中发挥着重要作用。我们的总体目标是了解细胞内血红素递送和插入胞浆蛋白的机制，以及如何在哺乳动物细胞中调节这一过程。我们发现一氧化氮 (NO) 会阻止细胞血红素插入一系列血红素蛋白（NO 合酶、细胞色素 P450、血红蛋白和过氧化氢酶）。利用这些信息以及诱导型一氧化氮合酶 (iNOS) 作为我们的模型血红素蛋白，我们发现甘油醛 3-磷酸脱氢酶 (GAPDH) 是该过程中的关键角色。我们的初步研究表明，NO 通过促进 GAPDH 的特异性 S 亚硝化来抑制细胞血红素插入 iNOS，从而改变与其酶活性无关的 GAPDH 特性。我们假设 GAPDH 通过其 S-亚硝化调节的非传统功能在细胞内血红素递送中发挥重要作用。我们提出细胞、生物化学和生物物理方法来测试我们一般假设的三个方面： 目标 1. GAPDH 在细胞内血红素输送和稳态中发挥一般作用吗？我们将通过确定 GAPDH 是否参与血红素插入四种蛋白质（血红蛋白、细胞色素 P450、组成型 NOS 和过氧化氢酶）以及 GAPDH 是否参与血红素递送至血红素加氧酶 1 和 2 来对此进行测试。目标 2. 细胞是否控制通过调节蛋白质 S-亚硝化能力来调节血红素插入反应？我们将通过以下方式对此进行测试：(i) 上调或下调已知细胞脱亚硝基酶（硫氧还蛋白-1 和 GSH-NO 还原酶）的表达水平，以改变响应 NO 的细胞 S-亚硝基蛋白（蛋白-SNO）的积累，然后 (ii) 确定这些变化是否会改变细胞中总蛋白 SNO 和 SNO-GAPDH 的水平，并以可预测的方式改变细胞血红素插入的 NO 敏感性。目标 3. GAPDH 的血红素结合、血红素转移和蛋白质相互作用特性与细胞血红素递送有何关系？我们将通过以下方式对此进行研究：(i) 测量纯 GAPDH 蛋白的血红素结合参数，这些参数在支持细胞中血红素插入的能力方面有所不同（野生型、S-亚硝化、点突变体），(ii) 评估 GAPDH 蛋白与脱辅基蛋白靶点及其在体外重构系统中转移结合血红素的能力，以及(iii)通过蛋白质晶体学和光谱学解析GAPDH-血红素复合物的结构。我们的研究将共同​​揭示 NO、蛋白质 S-亚硝化和 GAPDH 在细胞内血红素递送和插入蛋白质中的新作用。这将增进我们对细胞和分子水平基本过程的理解，并为研究这些方面如何在器官和整个动物水平影响人类健康和疾病奠定基础。 公共卫生相关性：血红素蛋白存在于我们的细胞中，并且在我们的身体中具有许多重要作用。我们的研究将填补关于血红素蛋白如何在我们的细胞中产生以及这个过程如何受到一氧化氮（一种由我们的身体自然产生的作为控制剂的气体）调节的知识的巨大空白。我们的研究结果将涉及人类健康和疾病的许多方面，包括血液形成、药物反应、贫血和炎症。