The role of GILT in the generation of reactive oxygen species

 描述（由申请人提供）：γ干扰素诱导型溶酶体硫醇还原酶（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。它在抗原呈递细胞 (APC) 中组成型高表达，在含有二硫键的抗原由 MHC II 类分子呈递给 CD4 T 细胞的过程中发挥着关键作用，在树突状细胞中，MHC I 类分子将抗原交叉呈递给 CD8 T 细胞。大多数细胞类型中的 GILT 也可由 γ 干扰素 (IFN-γ) 和其他炎症细胞因子诱导。 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加工中发挥作用之外，还可能具有其他功能。该提议基于我们对这样一个功能的发现：暴露于细菌脂多糖（LPS）或其他病原体相关分子模式（PAMP）的巨噬细胞需要GILT来最佳地产生呼吸爆发。缺乏酶活性 GILT 的巨噬细胞产生的活性氧 (ROS) 较少，并且杀死吞噬细菌（包括鼠伤寒沙门氏菌、大肠杆菌和金黄色葡萄球菌）的能力受损。 GILT基因被删除的小鼠清除鼠伤寒沙门氏菌腹膜感染的能力受损。我们建议确定这些现象的机制基础。我们将确定 GILT 是否影响巨噬细胞胞质的整体氧化还原电位。我们将确定 GILT 表达是否影响巨噬细胞和中性粒细胞中负责 ROS 生成的主导系统成分、NADPH 氧化酶-2 (Nox2) 的稳态细胞内分布，以及它是否影响在有或没有 IFN-γ 刺激的 LPS 刺激和吞噬作用下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体中作为 GILT 活性目标的分子，特别关注发光暴露的 Nox2 成分。我们还将确定 GILT 在其他细胞类型产生 ROS 中的潜在作用。其中包括使用 Nox2 的原型细胞类型、中性粒细胞以及可以使用 Nox2 同源物产生 ROS 的其他细胞类型。其中一些同源物与 Nox2 共享调控组件，但其他同源物则不然。这些研究将使我们能够确定 GILT 是否在 ROS 生成中发挥一般作用，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.