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，可控制多种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化在控制中性粒细胞肌动蛋白动力学中的作用。为了响应炎症刺激，中性粒细胞从循环血液迁移到感染组织，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是细胞过程如细胞粘附、迁移和吞噬作用的关键，因此对于中性粒细胞履行其在先天免疫中的作用至关重要。许多因素已牵连在信号诱导的肌动蛋白聚合，但潜在的负调制器的本质仍然知之甚少。我们最近报道，NADPH氧化酶依赖的生理产生的活性氧负调控肌动蛋白聚合刺激中性粒细胞通过驱动可逆的肌动蛋白谷胱甘肽。蛋白质谷胱甘肽化是一个动态的过程，去谷胱甘肽化受谷氧还蛋白1（Grx 1）的严格调控。通过过度表达Grx 1减少肌动蛋白谷胱甘肽化增加F-肌动蛋白水平，而通过破坏Grx 1升高肌动蛋白谷胱甘肽化降低F-肌动蛋白水平。因此，Grx 1的破坏损害了中性粒细胞的趋化性。此外，Grx 1缺陷的小鼠中性粒细胞表现出受损的体内招募到炎症部位和降低杀菌能力。总之，这些结果提出了一个新的生理作用，谷胱甘肽和ROS诱导的可逆肌动蛋白谷胱甘肽化在调节肌动蛋白动力学在中性粒细胞，使我们假设，ROS诱导的肌动蛋白谷胱甘肽化是一个关键的调节机制，控制中性粒细胞的先天免疫功能。我们以前的研究表明，肌动蛋白谷胱甘肽化在中性粒细胞趋化中起着关键作用，需要很好地调节。为了进一步了解ROS诱导的肌动蛋白谷胱甘肽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Grx 1和肌动蛋白谷胱甘肽化在调控肌动蛋白动力学的细胞过程中的贡献，而不是趋化性，包括粘附，吞噬，细菌杀伤，转向和信号转导（目的I）。此外，Grx 1在调节中性粒细胞在体内运输的作用将在小鼠提睾肌模型使用活体显微镜（目的II）进行研究。终于来了我们将直接阐明Grx 1在鼠大肠杆菌肺炎模型（Aim III）中的先天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).