INTERACTION OF RAP1A PROTEIN W/ NEUTROPHIL CYTOCHROME B

中性粒细胞在许多类型的炎症性疾病中起着关键作用， 包括慢性肺部疾病、动脉粥样硬化、类风湿和急性 炎症性关节炎。造成这些损害的主要因素之一 疾病似乎是中性粒细胞产生的超氧阴离子。 人中性粒细胞超氧化物歧化系统的激活 被认为涉及细胞色素b-559的相互作用或组装， 这一系统的末端组件，与其他胞浆和膜 蛋白质。在此之前，我们报道了与ras相关的 蛋白，Rap1a，与中性粒细胞细胞色素b-559结合(Quinn，M.T.，等人)。 (1989)自然342：198-200)。这种拟核苷的结合 结合细胞色素b-559的三磷酸(GTP)结合蛋白 该蛋白在细胞的结构和/或功能中的可能作用 细胞色素，因此，在调节中性粒细胞超氧化物 制作。为了解决这种可能性，拟议的研究将集中于 关于Rap1蛋白发挥作用的基本假设的研究 在细胞色素b-559功能中的作用，可能是一个分子开关 调节细胞色素b-559与细胞色素b-559的其他成分的相互作用 NADPH氧化酶系统。 具体地说，本提案描述了以下战略：1)定性 细胞色素b：Rap1a结合。这些络合物的稳定性 将检查关于盐、Ph、Quanine的影响 核苷酸和洗涤剂。2)中性粒细胞的作用分析 活化状态对其稳定性、数量和化学计量的影响 复合体。RAP1A在细胞发育过程中的磷酸化状态 激活也将被确定。3)结构的确定 Rap1a的基础：细胞色素b结合，包括测定 哪个细胞色素b亚基与Rap1a和 这种相互作用的区域。4)功能角色的确定 系统。5)Rap1a-细胞色素b结合的结构分析 评价Rap1a和细胞色素诱导的构象变化 结合和GTP的结合。分析将包括亲和力 层析、荧光光谱、傅里叶变换红外 光谱学和荧光能量转移分析构象 改变。这些研究的完成将为 了解Rap1a在细胞的结构和功能中的作用 细胞色素及其复合体在中性粒细胞超氧化物中的作用 发电系统。这种理解最终可能导致潜在的 减少炎症性疾病中超氧化物歧化的治疗策略 疾病。此外，这些研究将为 了解ras相关蛋白在细胞中的作用及其机制 可能与功能效应器系统相互作用。

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.