Nitric Oxide Signaling with Heme Proteins

血红素蛋白的一氧化氮信号传导

• 批准号: 7227879
• 负责人: WILLIAM R. MONTFORT
• 金额: $ 21.48万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7227879
• 关键词: Apoptosis; Area; Bedbugs; Binding; Blood; Blood coagulation; Carrier Proteins; Catalytic Domain; Chagas Disease; Chemicals; Chemistry; Complex; Crystallography; Cysteine; Cytochrome P450; Environment; Family; Free Radicals; Funding; Genes; Grant; Heme; Hemeproteins; Hemoglobin; Human; In Vitro; Injection of therapeutic agent; Insecta; Invaded; Kinetics; Length; Link; Literature; Mammalian Cell; Manduca sexta; Measurement; Mechanics; Medical; Memory; Modification; Molecular Conformation; Movement; Mutagenesis; Nature; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Play; Production; Protein Dynamics; Proteins; Rate; Rattus; Reaction; Recombinants; Regulation; Resolution; Rhodnius; Role; Saliva; Salivary Glands; Signal Transduction; Soluble Guanylate Cyclase; Spectrum Analysis; Stress; Structure; Sulfhydryl Compounds; System; Thermodynamics; Time; Tissues; Trypanosoma; Vasodilation; Water; computer studies; cytochrome c oxidase; feeding; genetic regulatory protein; heme a; in vivo; insight; killings; macrophage; mutant; nitrophorin; oxidation; protein function; quantum; research study; sucking; ultra high resolution

This proposal is the competitive renewal of grant HL62969. Our studies center on nitric oxide (NO) and its interactions with heme proteins, using a variety of protein examples and numerous, largely biophysical, approaches. NO regulates activities in all mammalian tissues and plays a central role in diverse areas, including vasodUation, blood coagulation, memory formation, oxygen stress, apoptosis, and macrophage killing of invading species. NO is produced by NO synthase and detected by soluble guanylate cyclase (sGC), both of which interact with NO through heme. Other molecules possibly influenced by NO include cytochrome oxidase, hemoglobin, and gene regulatory proteins with free thiols. We have entered this field through a family of insect herne proteins, called nitrophorins, that transport NO from the saliva of the insect to the tissues of potential victims, leading to vasodilation, reduced blood coagulation and a satisfying blood meal for the insect. We have expressed and characterized five nitrophorins, four from Rhodnius prolixus, the kissing bug, which transports the trypanosome for Chagas' disease, and one from Cimex lectularius, the bed bug. We have determined crystal structures of four nitrophodns, to as high as 0.85 A resolution, and have kinetic, spectroscopic, thermodynamic, mutagenic, theoretical and computational studies underway to link structure to function in NO transport. Thus studies have revealed heme molecules that are highly distorted (ruffled), which may serve to stabilize the ferric oxidation state required for nitrophorin function. They have also revealed a substantial NO-induced conformational change in the nitrophorins that apparently serves to desolvate and protect NO from reaction with water and oxygen. We are also extending our studies to other NO sensing proteins, including sGC. In the next funding period, we propose experiments to (1) understand the role of heme distortion in nitrosyl complex formation and signaling; (2) understand the role of protein conformational change in NO binding and release by the Rhodnius nitrophorins; (3) uncover the mechanism for NO transport by Cimex nitrophorin, which appears to involve reversible S-nitrosylation; and (4) produce and characterize recombinant sGC, a complicated protein of central importance to NO signaling. These studies will provide unprecedented insight into the complicated chemistry of NO signaling.

本提案是HL62969拨款的竞争性续期。我们的研究集中在一氧化氮（NO）及其与血红素蛋白的相互作用，使用各种蛋白质的例子和大量的，主要是生物物理方法。NO调节所有哺乳动物组织的活动，并在多个领域发挥核心作用，包括血管扩张、血液凝固、记忆形成、氧应激、细胞凋亡和巨噬细胞杀死入侵物种。NO由NO合成酶产生，由可溶性鸟苷酸环化酶（sGC）检测，两者通过血红素与NO相互作用。其他可能受NO影响的分子包括细胞色素氧化酶、血红蛋白和带有游离硫醇的基因调节蛋白。我们是通过一类昆虫体内的蛋白质进入这个领域的，这种蛋白质叫做硝基磷蛋白，它能从昆虫的唾液中运输一氧化氮