棕榈酰化影响NOD1/2调控炎症反应的重要机制

The NOD-like receptors (NLRs) NOD1 and NOD2 have key roles in regulating innate immune responses and their mutations are common risk factors in inflammatory bowel disease and other inflammatory conditions. They are cytosolic pattern-recognition receptors critical for detection of microbial products. Upon binding to their cognate ligands (γD-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP)), they trigger innate inflammatory responses followed by the engagement of adaptive immunity. NOD1/2 have been demonstrated to be associated with plasma membrane and bacterial containing endosomes and this association is well understood to have a key role in microbial products detection. However, little is known about NOD1/2 recruitment mechanisms. Our preliminary studies suggest that palmitoylation is required for NOD1 (and very likely NOD2) recruitment to plasma and bacterial containing endosomes, which we experimentally verified using a pharmacological approach by using 2BP, a broad palmitoyl incorporation inhibitor, and mutational analysis. Both 2BP and NOD1 palmitoylation deficient mutant, NOD1(C952S), prevented NOD1 recruitment to plasma membrane and bacterial containing endosome as assessed by confocal microscopy, inhibited NOD1 palmitoylation as assessed by Acyl-biotin exchange assays, and reduced NOD1 mediated signaling cascade as indicated by NF-κB luciferase reporter assays. In addition, through BioID (a novel mass spectrometry analysis to query near-neighbors), we identified ZDHHC5 as the putative NOD1 palmitoyl-transferase. Based on these promising preliminary experimental results, we intend not only to further understand the functional consequences of NOD1 palmitoylation but also to extend these studies to NOD2. In addition, we plan to investigate the role of ZDHHC5 in NOD1/2 mediated signaling and in inflammation. Our findings will provide a better understanding of NOD1/2 in innate immunity and facilitate advances in discovery of novel treatments for NOD-driven inflammatory diseases.

NOD1和NOD2（NOD1/2）是细胞质中抗细菌免疫的关键模式识别受体，在病原菌感染所致的炎症反应和自身免疫疾病的发生中有重要作用。研究表明NOD1/2行使功能时需要质膜及含病原菌内体膜上的定位。然而NOD1/2的定位调控机制及与其功能的关系尚不清楚。本项目组在前期研究中采用新的蛋白互作质谱联用法（BioID），酰基生物素置换法（ABE）及荧光素酶报告系统，发现NOD1的棕榈酰化修饰是影响其亚细胞定位及正确免疫应答功能的关键因素，并初步鉴定了NOD1棕榈酰化的发生位点及相应的棕榈酰转移酶ZDHHC5。本项目拟在此基础上进一步解析ZDHHC5依赖的棕榈酰化修饰及其调控NOD1/2免疫应答的机制。项目的顺利实施能够更深入理解NOD1/2介导细菌免疫应答的机理，不仅可在理论上丰富固有免疫系统的基础研究，更能为阐明相关疾病（如克罗恩病、炎症性肠病等）的致病机理及相应治疗药物的开发提供理论依据。

NOD样受体（NLR）家族成员NOD1和NOD2是细胞质中抗细菌免疫的关键模式识别受体，在病原菌感染所致的炎症反应和自身免疫疾病的发生中有重要作用。研究表明NOD1/2在细胞质膜及含病原菌内体膜上定位，识别他们相应的配体iE-DAP或MDP，从而起始炎症信号通路并进一步诱发适应性免疫。然而NOD1/2的定位调控机制及与其功能的关系尚不清楚。本项目组在前期的研究中使用广谱棕榈酰转移酶抑制剂2BP，结合定点突变的手段，首先明确了NOD1和NOD2的棕榈酰化修饰。同时，在该研究中运用新的蛋白互作质谱联用法（BioID），酰基生物素置换法（ABE）及荧光素酶报告系统，发现NOD1的棕榈酰化修饰是影响其亚细胞定位及正确免疫应答功能的关键因素，并初步鉴定了NOD1棕榈酰化的发生位点及相应的棕榈酰转移酶ZDHHC5。与此同时，本项目利用ZDHHC5条件性敲出小鼠鉴定了ZDHHC5催化NOD1和NOD2的棕榈酰化修饰，对小鼠巨噬细胞免疫应答及识别病原菌入侵具有重要作用。本项目的研究成果在临床上为遗传性肠炎提供潜在的新的诊断标志，未来有望通过设计新的治疗方案，缓解或者治愈炎性肠病，并解决炎性肠病中出现的耐药性问题。