REGULATION OF PHAGOCYTE FUNCTION

吞噬细胞功能的调节

• 批准号: 2071936
• 负责人: ULLA G. KNAUS
• 金额: $ 14.01万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2071936
• 关键词: NAD(P)H oxidoreductase; cell motility; enzyme activity; gene expression; guanine nucleotide binding protein; guanosine diphosphate; guanosine triphosphate; human subject; molecular cloning; monocyte; nucleic acid sequence; phagocytes; phagocytosis; protein purification; recombinant proteins; secretion; transfection

Human phagocytic white blood cells serve as a major cellular defense against bacterial infection. These cells undergo a complex series of responses, including chemotactic and motile responses, uptake of microorganisms by phagocytotic processes, and bacterial killing by secretion of granule contents and the generation of toxic oxygen metabolites. This highly organized and regulated series of events triggered by infectious agents can be understood at its basic level utilizing many of the same underlying mechanisms as in normal cellular processes. This includes the widespread involvement of GTP-binding proteins in phagocytic white blood cell activities. It has been shown that the NADPH oxidase, the site of superoxide production in human neutrophils, is regulated by the ras-related GTP- binding protein Rac2. The activity of this, and related GTP-binding proteins is determined by factors which regulate the binding of GTP (active) and GDP (inactive). Thus GTPase activating proteins (GAPs) stimulate GTP hydrolysis, while GDP dissociation stimulators (GDS) and GDP dissociation inhibitors (GDI) regulate nucleotide exchange. l propose to identify and investigate regulatory components for Rac2 which would determine the activity of the NADPH oxidase in response to infectious agents. In related studies, l will determine the role of Rac protein in regulating the cell biology of motility phagocytosis or bacterial uptake, and granule secretion. Assays for regulatory GDS and GAP proteins will be developed using recombinant Rac2, and these proteins will be identified in the human neutrophil. The proteins will be purified to homogeneity, sequenced, and cloned. We will then utilize these proteins to investigate the regulation of the NADPH oxidase. This system will provide a model to understand Rac regulation that will then be extended to other cellular systems in which Rac and related proteins are required. Methodologies for the transfection and expression of Rac, Rac mutants and regulatory proteins into HL-60 promyelocytic cells will be developed. Using dominant negative or constitutively active forms of Rac protein, we will determine whether Rac, or the related Rho protein, play critical roles in the cell biology of neutrophils and/or monocytes. The mechanisms by which other systems, crucial for bacterial uptake and killing, are regulated by Rac and Rac regulatory proteins will be investigated using our findings in the oxidase system as a working model. Such studies will lead to increased knowledge of the cell biology of human phagocytes at the molecular level, and to novel approaches in anti-infective therapy.

人类吞噬的白细胞是一种主要的细胞防御 防止细菌感染。这些细胞经历了一系列复杂的 反应，包括趋化反应和运动反应，摄取 吞噬过程中的微生物，以及细菌的杀灭 颗粒物的分泌与有毒氧的产生 代谢物。这是一系列高度组织和监管的活动 由感染性病原体触发的可以在基础水平上被理解 利用许多与正常细胞相同的潜在机制 流程。这包括GTP绑定的广泛参与 吞噬白细胞活动中的蛋白质。 已有研究表明，超氧化物的位置NADPH氧化酶 人类中性粒细胞的产生，受ras相关的GTP- 结合蛋白rac2。该酶的活性，以及相关的GTP结合 蛋白质是由调节GTP结合的因素决定的 (活跃)和GDP(非活跃)。因此GTP酶激活蛋白(GAP) 刺激GTP水解，而GDP解离刺激因子(GDS)和GDP 解离抑制物(GDI)调节核苷酸交换。L向 识别和调查rac2的调节组件，这些组件将 测定NADPH氧化酶活性对感染性疾病的反应 探员们。在相关研究中，L将确定Rac蛋白在 调节运动吞噬或细菌摄取的细胞生物学， 和颗粒分泌物。 调节性GDS和GAP蛋白的分析将使用 重组的rac2，这些蛋白将在人类体内得到鉴定 中性粒细胞。这些蛋白质将被提纯到均一，测序，并 克隆的。然后我们将利用这些蛋白质来研究这种调节。 NADPH氧化酶。该系统将提供一个了解RAC的模型 然后将扩展到其他蜂窝系统的监管 RAC和相关蛋白质是必需的。 Rac、Rac突变体和Rac基因的转染和表达方法 将开发进入HL-60早幼粒细胞的调节蛋白。 使用显性的负性或结构性活性形式的RAC蛋白，我们 将决定RAC或相关的Rho蛋白是否起关键作用 中性粒细胞和/或单核细胞在细胞生物学中的作用。其作用机制 对细菌的吸收和杀灭至关重要的其他系统通过这些系统 受RAC和RAC调节蛋白的调节将使用 我们在氧化酶系统中的发现作为一个工作模型。这样的研究将 增加了对人类吞噬细胞生物学的认识 分子水平，以及抗感染治疗的新方法。