The role of GILT in the generation of reactive oxygen species

 描述(申请人提供)：干扰素诱导的溶酶体硫醇还原酶(GILT)是内吞途径中唯一已知的可以还原蛋白质中二硫键的酶。它在抗原提呈细胞(APC)中呈结构性高表达，在含有二硫键的抗原的加工过程中起关键作用，由MHC II类分子呈递给CD4T细胞，在树突状细胞中，MHC I类分子将抗原交叉递呈给CD8T细胞。在大多数细胞类型中，干扰素(干扰素-)和其他炎性细胞因子也可诱导GILT。Gilt的同源物存在于许多缺乏适应性免疫系统的物种中，这表明它除了在抗原处理方面发挥作用外，还可能具有其他功能。这一建议是基于我们发现的一个这样的功能：GILT是接触细菌脂多糖(LPS)或其他病原体相关分子模式(PAMP)的巨噬细胞最佳产生呼吸爆发所必需的。缺乏酶活性镀金的巨噬细胞产生的活性氧(ROS)较少，并且其杀灭吞噬细菌的能力受到损害，包括鼠伤寒沙门氏菌、大肠杆菌和金黄色葡萄球菌。Gilt基因缺失的小鼠清除鼠伤寒沙门氏菌腹膜感染的能力受到了损害。我们建议确定这些现象的机械基础。我们将确定镀金是否影响巨噬细胞胞浆的总氧化还原电位。我们将确定GILT的表达是否影响巨噬细胞和中性粒细胞中负责产生ROS的主要系统的成分的稳态细胞内分布，NADPH氧化酶-2(NOX2)，以及它是否影响在有和没有干扰素-刺激的情况下，在脂多糖刺激和吞噬时观察到的这些成分的分布变化。我们还将确定巨噬细胞溶酶体和吞噬小体中的分子是GILT活性的目标，特别关注发光暴露的NOX2成分。我们还将通过其他类型的细胞来确定GILT在ROS生成中的潜在作用。这些细胞包括使用NOX2的典型细胞类型、中性粒细胞和其他类型的细胞，这些细胞可以使用NOX2的同系物产生ROS。其中一些同源物与NOX2具有共同的调控成分，但另一些则不同。这些研究将使我们能够确定Gilt是否在ROS的产生中发挥普遍作用，并确定其作用的分子机制。

 DESCRIPTION (provided by applicant): Gamma Interferon-inducible Lysosomal Thiolreductase (GILT) is the only known enzyme in the endocytic pathway that can reduce disulfide bonds in proteins. It is constitutively highly expressed in antigen presenting cells (APCs) and plays a key role in the processing of antigens containing disulfide bonds for presentation by MHC class II molecules to CD4+ T cells and, in dendritic cells, cross-presentation of antigens by MHC class I molecules to CD8+ T cells. GILT is also inducible by gamma interferon (IFN-) and other inflammatory cytokines in most cell types. Homologues of GILT are present in many species that lack an adaptive immune system, suggesting that it may have functions in addition to its role on antigen processing. This proposal is based on our discovery of one such function: GILT is required for the optimal generation of the respiratory burst by macrophages exposed to bacterial lipopolysaccharide (LPS) or other pathogen-associated molecular patterns (PAMPs). Macrophages lacking enzymatically active GILT produce less reactive oxygen species (ROS) and are impaired in their ability to kill phagocytosed bacteria, including Salmonella typhimurium, Escherichia coli and Staphylococcus aureus. Mice in which the GILT gene is deleted are impaired in their ability to clear a peritoneal infection by Salmonella typhimurium. We propose to determine the mechanistic underpinnings of these phenomena. We will determine whether GILT influences the overall redox potential of the macrophage cytosol. We will determine if GILT expression influences the steady state intracellular distribution of the components of the dominant system responsible for ROS generation in macrophages and neutrophils, NADPH oxidase- 2 (Nox2), and whether it affects the changes in distribution of these components observed upon LPS stimulation and phagocytosis, with and without IFN- stimulation. We will also determine the molecules in macrophage lysosomes and phagosomes that are targets for GILT activity, with a particular focus on Nox2 components that are luminally exposed. We will also determine the potential role of GILT in ROS generation by other cell types. These include the prototypical cell type that uses Nox2, the neutrophil, and other cell types that can generate ROS using homologues of Nox2. Some of these homologues share regulatory components with Nox2 but others do not. These studies will allow us to determine if GILT plays a general role in ROS generation, and to determine the molecular mechanism by which it acts.