INTERACTION OF RAP1A PROTEIN W/ NEUTROPHIL CYTOCHROME B

RAP1A 蛋白与中性粒细胞色素 B 的相互作用

• 批准号: 3457540
• 负责人: MARK T QUINN
• 金额: $ 10.91万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3457540
• 关键词: NAD(P)H dehydrogenase; acidity /alkalinity; affinity chromatography; antibody formation; cell free system; chemical association; chimeric proteins; conformation; crosslink; cytochrome b; cytoskeletal proteins; detergents; enzyme substrate complex; fluorescence spectrometry; guanine nucleotide binding protein; guanine nucleotides; high performance liquid chromatography; human subject; infrared spectrometry; laboratory rabbit; neutrophil; phospholipids; phosphorylation; posttranslational modifications; protein purification; protein reconstitution; protein sequence; protein structure function; sodium chloride; superoxides

Neutrophils play a key role in many types of inflammatory diseases, including chronic lung disease, atherosclerosis, and rheumatoid and acute inflammatory arthritis. One of the primary agents of damage in these diseases appears to be the neutrophil-generated superoxide anion. Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b-559, the terminal component of this system, with other cytosolic and membrane proteins. Previously, we reported the association of a ras-related protein, Rap1A, with neutrophil cytochrome b-559 (Quinn, M.T., et al. (1989) Nature 342: 198-200). The association of this quanosine triphosphate (GTP)-binding protein with cytochrome b-559 suggested a possible role for this protein in the structure and/or function of the cytochrome and, hence, in the regulation of neutrophil superoxide production. To address this possibility, the proposed studies will focus on investigating the fundamental hypothesis that the rap1 protein plays a role in the function of cytochrome b-559, possibly as a molecular switch that regulates the cytochrome b-559 interaction with other components of the NADPH oxidase system. Specifically, this proposal describes strategies for: 1) Characterization of the cytochrome b:Rap1A association. The stability of these complexes will be examined with respect to the effects of salts, Ph, quanine nucleotides and detergents. 2) Analysis of the effect of neutrophil activation state on the stability, quantity, and stoichiometry of the complexes. The phosphorylation state of Rap1A during stages of cell activation will also be determined. 3) Determination of the structural basis of the Rap1A:cytochrome b association, including a determination of which cytochrome b subunit associates with Rap1A and the sequences of the regions of this interaction. 4) Determination of the functional role of system. 5) Structural analysis of the Rap1A-cytochrome b association to evaluate conformational changes induced in Rap1A and cytochrome b y their association and by the binding of GTP. Analyses will include affinity chromatography, fluorescence spectroscopy, Fourier-transform infrared spectroscopy, and fluorescence energy transfer to analyze conformational changes. The accomplishment of these studies will provide a basis for the understanding of the role f Rap1A in the structure and function of the cytochrome and the role of this complex in the neutrophil superoxide generating system. This understanding may eventually lead to potential therapeutic strategies for reducing superoxide production in inflammatory disease. In addition, these studies will provide a basis for the understanding of the role of ras-related proteins in cells and their possible interaction with functional effector systems.

中性粒细胞在许多类型的炎症性疾病中起关键作用， 包括慢性肺部疾病，动脉粥样硬化以及类风湿和急性 炎症性关节炎。 这些损害的主要代理之一 疾病似乎是中性粒细胞生成的超氧化物阴离子。 人类嗜中性粒细胞中超氧化物生成系统的激活是 被认为涉及细胞色素B-559的相互作用或组装， 该系统的终端组件，以及其他胞质和膜 蛋白质。 以前，我们报告了与RAS相关的关联 蛋白质，Rap1a，中性粒细胞细胞色素B-559（Quinn，M.T。等。 （1989）自然342：198-200）。 Quanosine的关联 带有细胞色素B-559的三磷酸（GTP）结合蛋白表明 该蛋白在结构和/或功能中的可能作用 细胞色素，因此，在中性粒细胞超氧化物的调节中 生产。 为了解决这种可能性，拟议的研究将集中 研究RAP1蛋白发挥A的基本假设 在细胞色素B-559的功能中的作用，可能是分子开关 调节细胞色素B-559与其他组件的相互作用 NADPH氧化酶系统。 具体而言，该建议描述了：1）表征的策略 细胞色素B：RAP1A协会。 这些复合物的稳定性 将在盐，pH，喹氨酸的影响方面检查 核苷酸和洗涤剂。 2）分析中性粒细胞的作用 激活状态在稳定性，数量和化学计量学上的激活状态 复合物。 细胞阶段Rap1a的磷酸化状态 还将确定激活。 3）确定结构 RAP1A的基础：细胞色素B关联，包括确定 哪个细胞色素B亚基与Rap1a和序列相关联 这种相互作用的区域。 4）确定功能作用 系统。 5）RAP1A-CYTOCHROME B关联与 评估Rap1a和细胞色素诱导的构象变化。 关联和通过GTP的结合。 分析将包括亲和力 色谱，荧光光谱，傅立叶转换红外 光谱和荧光能量转移以分析构象 更改。 这些研究的完成将为 了解F Rap1a在结构和功能中的作用 细胞色素和该复合物在中性粒细胞超氧化物中的作用 生成系统。 这种理解最终可能导致潜力 减少炎症中超氧化物产生的治疗策略 疾病。 此外，这些研究将为 了解与RAS相关蛋白在细胞及其它们的作用 可能与功能效应器系统的相互作用。