REGULATION OF PHAGOCYTE FUNCTION

人类吞噬性白色血细胞作为主要的细胞防御 防止细菌感染。这些细胞经历一系列复杂的 反应，包括趋化和运动反应， 通过吞噬过程杀死微生物， 颗粒内容物的分泌和有毒氧气的产生 代谢物。 这一系列高度组织和规范的活动 可以从基本层面上理解 利用许多与正常细胞中相同的潜在机制， 流程.这包括GTP结合的广泛参与 吞噬白色血细胞活动中的蛋白质。 已经表明，NADPH氧化酶，超氧化物的位点， 在人类中性粒细胞中产生，由ras相关的GTP调节， 结合蛋白Rac 2。这种活性和相关的GTP结合 蛋白质是由调节GTP结合的因子决定的 （活跃）和国内生产总值（不活跃）。因此，GTP酶激活蛋白（GAP） 刺激GTP水解，而GDP解离刺激物（GDS）和GDP 解离抑制剂（GDI）调节核苷酸交换。我提议 确定并调查Rac 2的监管组件， 确定响应于感染的NADPH氧化酶的活性， 剂.在相关研究中，我将确定Rac蛋白在 调节运动性吞噬作用或细菌摄取的细胞生物学， 和颗粒分泌。 将使用以下方法开发用于调节GDS和GAP蛋白的测定： 重组Rac 2，这些蛋白将在人类中鉴定。 中性粒细胞。 将蛋白质纯化至同质，测序， 克隆的然后我们将利用这些蛋白质来研究调节 NADPH oxidase氧化酶。该系统将提供一个模型来理解Rac 然后将其扩展到其他蜂窝系统，其中 Rac和相关蛋白是必需的。 用于转染和表达Rac、Rac突变体和Rac突变体的方法 将开发用于HL-60早幼粒细胞的调节蛋白。 使用显性负性或组成型活性形式的Rac蛋白，我们 将决定Rac或相关的Rho蛋白是否起关键作用， 在嗜中性粒细胞和/或单核细胞的细胞生物学中的作用。的机制 其他对细菌吸收和杀死至关重要的系统， 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工作模型。这些研究将 导致人类吞噬细胞的细胞生物学知识的增加， 分子水平，以及抗感染治疗的新方法。

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.