New mechanism and regulation of intracellular heme delivery in mammals

描述（由申请人提供）：血红素蛋白在生理学和医学中起着重要作用。我们的主要目标是了解细胞内血红素的传递和插入到胞质蛋白的机制，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）阻断细胞血红素插入到一系列血红素蛋白（NO脱氢酶，细胞色素P450，血红蛋白和过氧化氢酶）。利用这些信息，和诱导型NO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）是一个关键的球员在这个过程中。我们的初步研究表明，NO通过促进GAPDH的特异性S-亚硝化作用抑制细胞血红素插入iNOS，从而改变GAPDH的特性，而不依赖于其酶活性。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起重要作用。我们提出了细胞，生物化学和生物物理的方法来测试我们的一般假设的三个方面：目的1。GAPDH在细胞内血红素的传递和稳态中是否起着普遍的作用？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。目标二。细胞是否通过调节蛋白质S-亚硝化的能力来控制血红素插入反应？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硝酶（硫氧还蛋白-1和GSH-NO还原酶）的表达水平，以改变细胞S-亚硝基蛋白（蛋白质-SNO）响应NO的积累，然后（ii）确定这些变化是否改变细胞中总蛋白质-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传递的关系如何？我们将通过以下方式对此进行调查：（i）测量纯GAPDH蛋白质的血红素结合参数，所述纯GAPDH蛋白质在其支持血红素插入细胞中的能力方面不同（野生型、S-亚硝化、点突变体），（ii）评估GAPDH蛋白质与脱辅基蛋白靶的相互作用及其在体外重构系统中转移结合的血红素的能力，和（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。总之，我们的研究将识别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.