Structure And Function Of Phagocyte Proteins

中性粒细胞和其他循环吞噬细胞在对感染或炎症刺激的反应中产生高水平的活性氧（ROS），这一过程被称为呼吸爆发。这种反应归因于产生超氧化物的NADPH氧化酶的活性，超氧化物是ROS的前体，ROS是重要的杀微生物剂和炎症介质。慢性肉芽肿病（CGD）患者NADPH氧化酶缺乏，对微生物感染和异常炎症反应的易感性增强。本项目探索吞噬细胞（phox系统）呼吸爆发氧化酶的细胞调节机制，并对非免疫细胞（Nox和Duox氧化酶）中表达的相关酶进行表征。我们描述了非髓系组织中活性氧的来源，特别是结肠、肾脏、甲状腺和唾液腺、粘膜表面、大脑和血管组织。在这些位点，氧化剂可以在宿主防御和炎症反应中起作用，或提供氧化还原信号，改变基因表达模式，介导对生长因子、分化、细胞衰老、细胞凋亡（程序性细胞死亡）或氧感应的增殖反应。在结肠氧化酶的研究中，我们检测了Nox1在结肠上皮细胞中的表达模式，并证明Nox1可由终末分化或干扰素- γ治疗诱导。Nox1在功能上取代gp91phox，在共表达细胞质因子p47phox和p67phox的细胞中恢复刺激依赖性超氧化物释放。此外，我们鉴定了这些细胞质因子（Noxo1 / p41和Noxo1 / p51）的独特的结肠特异性同源物，表明Nox1是一种受调节的phox样复合体，可能在宿主防御和结肠上皮炎症过程中起作用。我们正在比较几种细胞模型中可变剪接的Nox1亚型及其辅助因子的功能。相关工作是检查Nox1成分的亚细胞位置，并跟踪它们在细胞激活时的运动。在旨在探索肾氧化酶（Renox或Nox4）的功能作用的研究中，我们开发了Nox4表达增强或缺乏的转基因小鼠系。我们鉴定了四个缺失功能性Nox4基因的小鼠品系，这些品系被用来探索肾氧化酶在整个动物中的作用。目前的焦点是Nox4在肾氧感应和红细胞生成中的作用，因为Nox4水平似乎对缺氧和ROS有反应，被认为提供调节肾促红细胞生成素合成的负反馈信号。肾氧化酶似乎是一种组成活性酶，与它作为氧感应酶的作用一致。相关研究正试图鉴定支持Nox4催化核心的其他功能成分，如Rac、p22phox和细胞质phox样蛋白。最后，我们观察到甲状腺双氧化酶（Duox1和Duox2）在气道（气管、支气管）、唾液腺导管和直肠的上皮表面的功能性表达，表明这些酶是过氧化氢的来源，支持粘膜表面乳过氧化物酶的抗微生物活性。原代培养的人气道（支气管）和猴子唾液腺上皮细胞在触发钙释放的生理激动剂作用下，以双依赖（反义抑制）的方式产生过氧化氢。这些系统的开发是为了探索Duox表达与上皮细胞分化的关系，并确认这些氧化酶在粘膜表面的抗微生物防御和炎症过程中的作用。

Neutrophils and other circulating phagocytes generate high levels of reactive oxygen species (ROS) in response infectious or inflammatory stimuli, in a process known as the respiratory burst. This response is attributed to the activity of NADPH oxidase that produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) have NADPH oxidase deficiencies and suffer from enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the respiratory burst oxidase in phagocytes (phox system) and is characterizing related enzymes expressed in non-immune cells (Nox and Duox oxidases). We are characterizing sources of reactive oxygen species in non-myeloid tissues, notably colon, kidney, thyroid and salivary glands, mucosal surfaces, brain, and vascular tissue. In these sites, the oxidants can serve in host defense and inflammatory reactions or provide redox signals that alter gene expression patterns that mediate proliferation responses to growth factors, differentiation, cellular senescence, apoptosis (programmed cell death), or oxygen sensing. In studies on the colon oxidase, we examined expression patterns of Nox1 in colon epithelial cells and demonstrated that Nox1 is induced by terminal differentiation or by interferon-gamma treatment. Nox1 functionally replaces gp91phox, restoring stimulus-dependent superoxide release in cells co-expressing the cytosol factors p47phox and p67phox. Furthermore, we identified unique, colon-specific homologues of these cytosolic factors (Noxo1 / p41 and Noxa1 / p51), showing that Nox1 is a regulated, phox-like complex that may act in host defense and inflammatory processes in the colon epithelium. We are comparing the functions of variably spliced isoforms of Nox1 and its co-factors in several cell models. Related work is examining the sub-cellular location of Nox1 components and tracking their movement in response to cellular activation. In studies aimed at exploring the functional role of the renal oxidase (Renox or Nox4), we developed transgenic mouse lines with enhanced or deficient expression of Nox4. We identified four mouse strains in which the functional Nox4 gene is absent, which are being used to explore roles of the renal oxidase in whole animals. A current focus is on the proposed role of Nox4 in renal oxygen sensing and erythropoiesis, since Nox4 levels appear to respond to hypoxia and ROS are thought to provide negative feedback signals regulating renal erythropoietin synthesis. The renal oxidase appears to be a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. Related studies are attempting to identify other functional components supporting the catalytic core of Nox4, such as Rac, p22phox, and cytosolic phox-like proteins. Finally, we have observed functional expression of thyroid dual oxidases (Duox1 and Duox2) on epithelial surfaces of airways (trachea, bronchium), salivary gland ducts, and the rectum, suggesting these enzymes serve as sources of hydrogen peroxide supporting the anti-microbial activity of lactoperoxidase on mucosal surfaces. Primary cultured human airway (bronchial) and monkey salivary gland epithelial cells were shown to produce hydrogen peroxide in a Duox-dependent (antisense-inhibited) manner in response to physiological agonists that trigger calcium release. These systems are being developed to explore Duox expression in relation to epithelial cell differentiation and to confirm roles of these oxidases in anti-microbial defenses and inflammatory processes on mucosal surfaces.