New mechanism and regulation of intracellular heme delivery in mammals

描述(申请人提供)：血红素蛋白在生理学和医学中起着重要作用。我们的广泛目标是了解细胞内血红素输送和插入胞浆蛋白的机制，以及这一过程在哺乳动物细胞中是如何调节的。我们发现，一氧化氮(NO)阻止细胞内的血红素插入一系列血红素蛋白(没有合成酶，细胞色素P450‘S，血红蛋白和过氧化氢酶)。利用这一信息，并将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)在这一过程中起着关键作用。我们的初步研究表明，NO通过促进谷氨酸脱氢酶的S亚硝化，从而改变谷氨酸脱氢酶的性质，从而抑制细胞内血红素插入诱导型一氧化氮合酶。我们推测，谷氨酸脱氢酶通过受其S亚硝化调节的非传统功能，在细胞内的血红素转运中发挥重要作用。我们提出了细胞、生化和生物物理方法来检验我们的一般假设的三个方面：目的1.GAPDH在细胞内的血红素输送和动态平衡中起普遍作用吗？我们将通过确定谷氨酸脱氢酶是否参与血红素插入四种蛋白质(血红蛋白、细胞色素P450、构成一氧化氮合酶和过氧化氢酶)以及是否参与血红素向血红素加氧酶1和2的输送来测试这一点。目的2.细胞是否通过调节它们对蛋白质S亚硝化的能力来控制它们的血红素插入反应？我们将通过以下方法来测试这一点：(I)上调或下调已知的细胞脱氮酶酶(硫氧还蛋白-1和谷胱甘肽-NO还原酶)的表达水平，以改变细胞内S亚硝酸蛋白(蛋白质-SNO)的积聚，以响应NO，然后(Ii)确定这些变化是否改变细胞中总蛋白-SNO和SNO-GAPDH的水平，并以可预测的方式改变细胞对血红素插入的NO敏感性。目的3.谷氨酸脱氢酶的血红素结合、血红素转移和蛋白质相互作用特性与细胞内的血红素转运有何关系？我们将通过以下方式进行研究：(I)测量不同支持细胞内血红素插入能力的纯GAPDH蛋白(野生型、S亚硝化、点突变)的血红素结合参数；(Ii)评估GAPDH蛋白与载脂蛋白靶标的相互作用以及它们在体外重组系统中转移结合血红素的能力；(Iii)通过蛋白质结晶学和光谱学解决GAPDHH-血红素复合体的结构。总之，我们的研究将发现NO、蛋白质S亚硝化和谷氨酸脱氢酶在细胞内血红素运输和插入蛋白质中的新角色。这将促进我们对细胞和分子水平上的基本过程的理解，并将为研究这些方面如何在器官和整个动物水平上影响人类健康和疾病奠定基础。

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.