INTERACTION OF RAP1A PROTEIN W/ NEUTROPHIL CYTOCHROME B

中性粒细胞在许多类型的炎性疾病中起关键作用， 包括慢性肺病、动脉粥样硬化、类风湿性和急性 炎性关节炎 其中造成损害的主要因素之一 疾病似乎是嗜热菌产生的超氧阴离子。 人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， 该系统末端组分，以及其它胞质和膜组分 proteins. 以前，我们报道了ras相关的 蛋白Rap 1A与嗜中性粒细胞色素B-559（Quinn，M.T.，等人 （1989）Nature 342：198-200）。 这种喹核苷的结合 三磷酸（GTP）结合蛋白与细胞色素B-559的结合表明， 这种蛋白质在结构和/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 生产 为了解决这一可能性，拟议的研究将侧重于 在研究RAP 1蛋白质发挥作用的基本假设时， 在细胞色素B-559功能中的作用，可能作为分子开关 调节细胞色素B-559与其他成分的相互作用， NADPH氧化酶系统。 具体而言，该提案描述了以下策略：1）表征 细胞色素B：Rap 1A的结合。 这些配合物的稳定性 将检查盐，Ph，奎宁的影响 核苷酸和去污剂。 2)中性粒细胞的作用分析 活化状态的稳定性，数量和化学计量的 配合物 Rap 1A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 Rap 1A：细胞色素B结合的基础，包括确定 其中细胞色素B亚基与Rap 1A结合，并且细胞色素B亚基的序列与Rap 1A结合。 这种互动的区域。 4)职能作用的确定 系统 5)Rap 1A-细胞色素B结合的结构分析 评价Rap 1A和细胞色素B诱导的构象变化， 与GTP的结合。 分析将包括亲和力 色谱法，荧光光谱法，傅里叶变换红外光谱法 光谱和荧光能量转移来分析构象 变化 这些研究的完成，将为今后的 理解f Rap 1A在结构和功能中的作用， 细胞色素及其在中性粒细胞超氧化物中的作用 发电系统 这种理解可能最终导致潜在的 减少炎性组织中超氧化物产生的治疗策略 疾病 此外，这些研究将为 理解ras相关蛋白在细胞中的作用及其 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.