The role of N-chlorination, a novel oxidative post-translational modification, in host-pathogen interactions and chronic inflammation

次氯酸是一种剧毒的氧化剂，由免疫细胞在所谓的氧化爆发过程中产生。为了在与产生HOCl的免疫细胞的接触中生存下来，细菌进化出了许多针对这种抗菌化合物的防御机制。2014年，我们发现RIDA是一种细菌氧化还原调节的伴侣蛋白，有助于大肠杆菌在HOCl暴露下存活。这个伴侣被N-氯化激活，N-氯化是一种新的HOCl2诱导的翻译后修饰。我们还发现，通过硫氧还蛋白体系依赖于NADPH的还原，这种N-氯化反应是完全可逆的。从那时起，我们和其他人发现了更多的蛋白质，在接触HOCl、一些细菌(如cnox)和其他人类起源的蛋白质(如血清白蛋白和其他血浆蛋白)时，会发生N-氯化。这些N-氯化蛋白质已被证明既有免疫调节作用，也有保护作用。然而，到目前为止，这些发现大多是基于体外观察。因此，这种修饰在宿主-病原体相互作用和潜在的慢性炎症中的调节作用只是暗示的。在这里，我们建议开发化学工具，这将使我们第一次能够研究体内N-氯化的(病理)生理作用。首先，我们计划对硫氧还蛋白还原蛋白质N-氯胺进行生化表征，以确定N-氯化反应与硫醇-二硫键动态平衡相关的分子机制。这将揭示N-氯化作为一种调节修饰在宿主-病原体相互作用和慢性炎症中整合的基础。同时，我们打算合成一组探针，用于蛋白质N-氯胺的检测和定量。我们计划根据我们以前在定量氧化还原蛋白质组学方面的经验，表征这些探针，并开发一种化学蛋白质组学方法来量化体内蛋白质N-氯化的程度。利用这些工具，我们将能够确定硫氧还蛋白和谷胱甘肽系统在暴露于HOCl和免疫细胞的大肠杆菌中的细胞N-氯胺稳态中所起的作用。此外，我们将首次尝试检测幼年特发性关节炎患者样本中的蛋白质N-氯化，确定这种新的翻译后修饰在严重慢性炎症下的临床相关性。

Hypochlorous acid (HOCl), produced by immune cells during the so-called oxidative burst, is a highly toxic oxidant. To survive their encounter with HOCl-producing immune cells, bacteria have evolved numerous defense mechanisms against this antibacterial compound. In 2014, we found that RidA is a bacterial redox-regulated chaperone that helps Escherichia coli to survive HOCl exposure. This chaperone is activated by N-chlorination, a novel HOCl-induced post-translational modification. We also found that this N-chlorination is fully reversible through NADPH-dependent reduction by the thioredoxin system. Since then, we and others have discovered more proteins that undergo N-chlorination upon exposure to HOCl, some of bacterial (like CnoX) and others of human origin (like serum albumin and other plasma proteins). These N-chlorinated proteins have been shown to have both immunomodulatory as well as protective properties. However, until now, most of these findings are based on in vitro observations. A regulatory role in host-pathogen interactions and potentially chronic inflammation of this modification is, thus, only implied. Here we propose to develop the chemical tools that will allow us, for the first time, to study the (patho-)physiological role of N-chlorination in vivo, too. At first, we plan to biochemically characterize protein N-chloramine reduction by thioredoxin in order to identify the molecular mechanism that links N-chlorination to the thiol-disulfide homeostasis. This will reveal the underpinnings of the integration of N-chlorination, as a regulatory modification, in host-pathogen interactions and chronic inflammation. In parallel, we intend to synthesize a set of probes for the detection and quantification of protein N-chloramines. We plan to characterize these probes and develop a chemoproteomic methodology to quantify the extent of protein N-chlorination in vivo, based on our prior experience in quantitative redox proteomics. With these tools we will then be able to determine the role of the thioredoxin and glutathione system in cellular N-chloramine homeostasis in E. coli exposed to HOCl and immune cells. Additionally, we will, for the first time, attempt to detect protein N-chlorination in samples from patients suffering from juvenile idiopathic arthritis, determining the clinical relevance of this novel post translational modification under severe chronic inflammation.