Regulation of neutrophil function by ROS-induced actin glutathionylation

描述(由申请人提供)：翻译后修饰是一种基本的生物调节机制，它控制着广泛的蛋白质的位置和活性。本研究的目的是阐明活性氧(ROS)诱导的肌动蛋白谷胱甘肽基化在控制中性粒细胞肌动蛋白动力学中的作用。作为对炎症刺激的反应，中性粒细胞从循环血液迁移到受感染的组织，在那里它们通过吞噬、杀死和消化细菌和真菌病原体来保护宿主。肌动蛋白动力学的调节对细胞黏附、迁移和吞噬等细胞过程至关重要，因此对中性粒细胞发挥其在先天免疫中的作用至关重要。许多因素与信号诱导的肌动蛋白聚合有关，但潜在的负调制子的本质仍然知之甚少。我们最近报道，依赖NADPH氧化酶的生理产生的ROS通过驱动可逆的肌动蛋白谷胱甘肽基化，对刺激的中性粒细胞中的肌动蛋白聚合进行负性调节。蛋白质谷胱甘肽基化是一个动态过程，谷氧还蛋白1(Grx1)对谷胱甘肽脱硫基化有严格的调控作用。过表达Grx1使肌动蛋白谷胱甘肽水平降低，使F-肌动蛋白水平升高，而通过干扰Grx1使肌动蛋白谷胱甘肽水平升高则降低F-肌动蛋白水平。一直以来，Grx1的干扰都会损害中性粒细胞的趋化作用。此外，Grx1缺乏的小鼠中性粒细胞在体内对炎症部位的募集受到损害，杀菌能力降低。综上所述，这些结果提出了谷氧还蛋白和ROS诱导的可逆的肌动蛋白谷胱甘肽基化在调节中性粒细胞肌动蛋白动力学中的一个新的生理作用，从而使我们假设ROS诱导的肌动蛋白谷胱甘肽基化是控制天然免疫中性粒细胞功能的关键调控机制。我们先前的研究表明，肌动蛋白谷胱甘肽基化在中性粒细胞趋化中起着关键作用，需要很好地进行调控。为了进一步了解ROS诱导的肌动蛋白谷胱甘肽基化在感染和炎症过程中中性粒细胞募集和功能中的作用，我们将继续阐明Grx1和肌动蛋白谷胱甘肽基化在调控趋化以外的细胞过程中的作用，包括黏附、吞噬、细菌杀伤、转向和信号转导(Aim I)。此外，Grx1在调节体内中性粒细胞运输中的作用将在小鼠提睾肌模型中使用活体显微镜(AIM II)进行研究。终于来了。我们将直接阐明Grx1在小鼠大肠杆菌肺炎模型(AIM III)中的先天性免疫和宿主防御中的作用。

DESCRIPTION (provided by applicant): Post-translational modification is a fundamental biological regulatory mechanism that controls the location and activity of a wide range of proteins. The goal of this proposed study is to elucidate the role of reactive oxygen species (ROS)-induced actin glutathionylation in controlling actin dynamics in neutrophils. In response to inflammatory stimuli, neutrophils migrate from the circulating blood to infected tissues, where they protect their host by phagocytosing, killing, and digesting bacterial and fungal pathogens. The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators is still poorly understood. We recently reported that NADPH oxidase-dependent physiologically generated ROS negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Protein glutathionylation is a dynamic process and the deglutathionylation is tightly regulated by glutaredoxin 1 (Grx1). Reducing actin glutathionylation by over-expressing Grx1 increased F-actin level, while elevating actin glutathionylation by disrupting Grx1 decreased F-actin level. Consistently, disruption of Grx1 impaired neutrophil chemotaxis. Moreover, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a novel physiological role for glutaredoxi and ROS-induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils, leading us to hypothesize that ROS-induced actin glutathionylation is a key regulatory mechanism that controls neutrophil function in innate immunity. Our previous study demonstrates that actin glutathionylation plays a critical role and needs to be well regulated in neutrophil chemotaxis. To further understand the role of ROS-induced actin glutathionylation in neutrophil recruitment and function during infection and inflammation, we will continue to elucidate the contribution of Grx1 and actin glutathionylation in regulating actin dynamics in cellular processes other than chemotaxis, including adhesion, phagocytosis, bacterial killing, turning, and signal transduction (Aim I). Moreover, the role of Grx1 in regulating neutrophil trafficking in vivo will be investigated in a murine cremaster muscle model using intravital microscopy (Aim II). Finally. we will directly elucidate the role of Grx1 in innate immunity and host defense in a murine Escherichia coli pneumonia model (Aim III).