BIOCHEMISTRY OF MACROPHAGE NITRIC OXIDE SYNTHESIS

巨噬细胞一氧化氮合成的生物化学

• 批准号: 2654070
• 负责人: DENNIS J STUEHR
• 金额: $ 23.87万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2654070
• 关键词: Raman spectrometry; active sites; arginine; circular dichroism; cofactor; conformation; crystallization; dimer; electron spin resonance spectroscopy; electron transport; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; flavins; heme; hemoprotein; macrophage; nitric oxide; nitric oxide synthase; pteridines; reduction; site directed mutagenesis; tetrahydrobiopterin; ultraviolet spectrometry

Nitric oxide (NO) biosynthesis has emerged as an important factor in human health and diseae. NO generated in small amounts appears to serve a signaling facedown, whereas production of excessive amounts is linked to numerous diseases including sepsis, immune-type diabetes, inflammatory bowel disease, rheumatoid arthritis, carcinogenesis, multiple sclerosis, and transplant rejection. Immunostimulation induces expression of a distinct NO synthase isoform (iNOS) in many tissues, including lung, liver, kidney, heart, smooth muscle, and intestine. Because iNOS generates large amounts of NO, its expression has become increasingly linked to the diseases noted above. We are studying the biochemistry of mouse macrophage iNOS, which is highly homologous to human iNOS. Macrophage iNOS is a bi-domain enzyme that contains FAD, FMN, tetrahydrobiopterin, heme, and calmodulin. In macrophages and other cells, iNOS is expressed as a mixture of monomers and dimers, with the monomer being inactive regarding NO synthesis. We hypothesize that dimerization of iNOS is a key determinant in its activation, and may serve as a physiologic and/or pharmacologic control point for iNOS function within cells. We will test this hypothesis with four specific experimental objectives; First, we will determine if dimerization physically alters the heme environment, thus affecting its reactivity, or if dimerization unmasks binding sites for H4biopterin and L- arginine in the oxygenase domain, thus creating the enzyme active site. Techniques include a variety of spectroscopies, redox potentiometry, and radioligand binding. Secondly, we will investigate whether dimerization enables the iNOS reductase and oxygenase domains to communicate electronically in a productive manner. Techniques include visible spectroscopy and creation of iNOS heterodimeric structures composed of non- identical subunits that differ with regard to domain composition or amino acid sequence. Thirdly, we will seek to identify the specific protein regions involved in subunit dimeric interaction and cofactor binding within the iNOS oxygenase domain. This will involve testing oxygenase domain peptides and fragments for H4biopterin, heme, and L-arginine binding, determining their ability to form dimers, or antagonize dimerization; mapping the oxygenase domain with monoclonal antibodies raised against iNOS; examining oxygenase domain mutants for dimerization, cofactor binding, and catalytic function; and crystallization of the dimeric oxygenase domain. Lastly, we will investigate whether iNOS subunit dimerization is an important physiologic or pharmacologic control point in cells, by monitoring dimer formation in cells over time, relating it to levels of cellular factors thought to promote dimerization, and testing whether dimerization can be manipulated pharmacologically. Together, this will provide a comprehensive picture regarding how dimer formation activates iNOS, and how it can be controlled.

一氧化氮（NO）生物合成已成为人类的重要因素 健康与疾病。 少量产生似乎没有 信号面对家，而产量过多的产生与 许多疾病，包括败血症，免疫型糖尿病，炎症 肠病，类风湿关节炎，癌变，多发性硬化症， 和移植排斥。 免疫刺激诱导A的表达 在包括肺，肝脏，包括肺，肝，包括肺， 肾脏，心脏，平滑肌和肠。 因为iNOS会产生大 数量不超过，其表达越来越多地与 上面指出的疾病。 我们正在研究小鼠巨噬细胞iNOS的生物化学，这是高度的 与人iNOS同源。 巨噬细胞iNOS是一种双域酶 包含FAD，FMN，四氢无生蛋白，血红素和钙调蛋白。 在 巨噬细胞和其他细胞，iNOS表示为单体的混合物， 二聚体，单体对无合成无效。 我们 假设iNOS的二聚化是其关键决定因素 激活，可以用作生理和/或药理控制 iNOS功能在细胞内的点。 我们将用 四个特定的实验目标； 首先，我们将确定是否 二聚化在物理上改变了血红素的环境，从而影响了它的环境 反应性，或者如果二聚化揭示了H4biopterin和l-的结合位点 氧合酶结构域中的精氨酸，从而产生酶的活性位点。 技术包括各种光谱学，氧化还原电位法和 放射性结合。 其次，我们将调查是否二聚化 启用iNOS还原酶和氧合酶结构域进行通信 以富有成效的方式进行电子方式。 技术包括可见的 光谱和iNOS异二聚体结构的创建 与域组成或氨基不同的相同亚基 酸序列。 第三，我们将寻求确定特定蛋白质 与亚基二聚体相互作用和辅因子结合的区域 iNOS氧合酶结构域。 这将涉及测试氧合酶结构域 H4biopterin，血红素和L-精氨酸结合的肽和片段， 确定它们形成二聚体或拮抗二聚体的能力； 用反向的单克隆抗体映射氧合酶结构域 iNos;检查氧合酶结构域突变体以二聚化，辅因子 结合和催化功能；和二聚体的结晶 氧合酶结构域。 最后，我们将调查iNOS亚基是否 二聚化是重要的生理或药理控制点 通过监视细胞中的二聚体形成，随着时间的流逝，将其与 被认为促进二聚化和测试的细胞因素水平 是否可以通过药理操纵二聚化。 一起，这个 将提供有关二聚体如何形成的全面图片 激活iNOS及其如何控制。