The role of GILT in the generation of reactive oxygen species

 描述（由申请方提供）：γ干扰素诱导型溶酶体巯基还原酶（GILT）是胞吞途径中唯一已知的可还原蛋白质中二硫键的酶。它在抗原呈递细胞（APC）中组成型高度表达，并在处理含有二硫键的抗原以由MHC II类分子呈递给CD 4 + T细胞和在树突状细胞中由MHC I类分子交叉呈递抗原给CD 8 + T细胞中起关键作用。在大多数细胞类型中，GILT也可由γ干扰素（IFN-γ）和其他炎性细胞因子诱导。GILT的同源物存在于许多缺乏适应性免疫系统的物种中，这表明它可能除了在抗原加工中的作用之外还具有功能。该建议是基于我们发现的一个这样的功能：GILT是暴露于细菌脂多糖（LPS）或其他病原体相关分子模式（PAMP）的巨噬细胞呼吸爆发的最佳生成所必需的。缺乏酶活性GILT的巨噬细胞产生较少的活性氧（ROS），并且其杀死吞噬细菌（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基因缺失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械基础。我们将确定GILT是否影响巨噬细胞胞质溶胶的整体氧化还原电位。我们将确定GILT表达是否影响负责巨噬细胞和嗜中性粒细胞中ROS产生的主导系统组分NADPH氧化酶-2（Nox 2）的稳态细胞内分布，以及它是否影响在LPS刺激和吞噬作用（有和没有IFN-γ刺激）后观察到的这些组分分布的变化。我们还将确定巨噬细胞溶酶体和吞噬体中的分子，这些分子是GILT活性的靶点，特别关注发光暴露的Nox 2组分。我们还将确定GILT在其他细胞类型的ROS生成中的潜在作用。这些包括使用Nox 2的原型细胞类型、嗜中性粒细胞和可以使用Nox 2的同源物产生ROS的其他细胞类型。这些同源物中的一些与Nox 2共享调控组分，但其他同源物不共享。这些研究将使我们能够确定GILT是否在ROS产生中发挥一般作用，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.