New mechanism and regulation of intracellular heme delivery in mammals

描述（申请人提供）：血红素蛋白在生理和医学上发挥着重要作用。我们的主要目标是了解细胞内血红素传递和插入细胞质蛋白的机制，以及这一过程如何在哺乳动物细胞中受到调节。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合成酶、细胞色素P450’s、血红蛋白和过氧化氢酶）。利用这些信息，并将诱导NO合成酶（iNOS）作为模型血红素蛋白，我们发现甘油醛3-磷酸脱氢酶（GAPDH）在这一过程中起关键作用。我们的初步研究表明，NO通过促进GAPDH的特异性s -亚硝化抑制细胞血红素插入iNOS，从而改变GAPDH独立于其酶活性的特性。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、生化和生物物理方法来检验我们一般假设的三个方面：目的1。GAPDH是否在细胞内血红素传递和体内平衡中起一般作用？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是否参与血红素向血红素加氧酶1和2的传递来验证这一点。目标2。细胞是否通过调节蛋白质s -亚硝化能力来控制血红素插入反应？我们将通过：(i)上调或下调已知细胞脱硝基酶（硫氧还蛋白-1和GSH-NO还原酶）的表达水平来改变细胞中s -亚硝基蛋白（蛋白质- sno）的积累，以响应NO，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有何关系？我们将通过以下方式进行研究：(i)测量纯GAPDH蛋白的血红素结合参数，这些蛋白支持细胞中血红素插入的能力不同（野生型，s -亚硝化，点突变体）；（ii）评估GAPDH蛋白与载脂蛋白靶蛋白的相互作用及其在体外重建系统中转移结合血红素的能力；（iii）通过蛋白质晶体学和光谱分析GAPDH-血红素复合物的结构。总之，我们的研究将发现NO、蛋白质s -亚硝化和GAPDH在细胞内血红素传递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.