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: 8636030
• 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 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636030
• 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; biophysical techniques; catalase; enzyme activity; heme oxygenase-1; human NOS2A protein; mutant; nitric oxide reductase; novel; protein complex; public health relevance; 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.

描述（由申请人提供）：血红素蛋白在生理和医学中起重要作用。我们的广泛目标是了解细胞内血红素递送和插入胞质蛋白的机制，以及在哺乳动物细胞中如何调节该过程。我们发现一氧化氮（NO）阻止了细胞血红素插入到多种血红素蛋白（无合酶，细胞色素P450，血红蛋白和过催化酶）中。使用这些信息，以及可诱导的NO合酶（INOS）作为模型血红素蛋白，我们发现3-磷酸甘油醛脱氢酶（GAPDH）是该过程的关键参与者。我们的初步研究表明，NO通过促进GAPDH的特异性S-硝化作用抑制细胞血红素插入INOS，从而改变了与其酶活性无关的GAPDH性质。我们假设GAPDH通过其S-硝化调节的非传统功能在细胞内血红素递送中起着至关重要的作用。我们提出了细胞，生化和生物物理方法来检验我们一般假设的三个方面：目标1。GAPDH在细胞内血红素递送和体内稳态中是否起一般作用？我们将通过确定GAPDH是否参与血红素插入四种蛋白（血红蛋白，细胞色素P450，组成型NOS和过氧化氢酶），以及GAPDH是否参与血红素氧化酶1＆2的血红素递送1和2。AIM 2。DO通过调节其蛋白质的能力来测试。我们将通过：（i）抬高或下调已知细胞非致命酶酶的表达水平（硫氧还蛋白-1和GSH-NO还原酶）以改变细胞s-硝基蛋白（蛋白质-SNO）的堆积，以响应NO，然后（ii）确定这些细胞的细胞和SNO-SNO-sno和SNO-SNO-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-sno-gapd and in e sno-sno-gapd and the Ens and sno-sno-gapd的变化是否会改变。以可预测的方式插入。目标3。GAPDH的血红素结合，血红素转移和蛋白质相互作用与细胞血红素递送有关？我们将通过：（i）测量纯GAPDH蛋白的血红素结合参数，这些蛋白的血红素结合参数在支持细胞中的血红素插入能力上有所不同（野生型，s-硝化，点突变体），（ii）评估GAPDH蛋白与APO蛋白与APO蛋白与APO蛋白与APO蛋白质的相互作用及其在II II II II II II II II II II II II（II）中的相互作用（通过蛋白质晶体学和光谱法进行GAPDH血清复合物。总之，我们的研究将辨别NO的新作用，蛋白质S-硝化作用以及GAPDH在细胞内血红素递送和插入蛋白质中的作用。这将使我们在细胞和分子水平上对基本过程的理解，并将为研究这些方面如何影响器官和整个动物水平的人类健康和疾病。