Characterization of the NOD2 NBD domain and role in chronic inflammation

NOD2 NBD 结构域的表征及其在慢性炎症中的作用

• 批准号: 8795675
• 负责人: Jae W Dugan
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795675
• 关键词: APAF1 gene; ATP Hydrolysis; ATP phosphohydrolase; Acetylmuramyl-Alanyl-Isoglutamine; Agonist; Amino Acid Sequence; Animal Model; Antibiotic Resistance; Arginine; Arthritis; Asthma; Autophagocytosis; Autophagosome; Bacteria; Bacteriology; Binding; Binding Sites; Biochemical; Bone Marrow; C-terminal; Caspase; Cell Wall; Cells; Chlamydia; Chronic; Clinical; Code; Crohn' s disease; Data; Defect; Development; Disease; Doctor of Medicine; Doctor of Philosophy; Etiology; Event; Exposure to; Eye; Family; Family member; Future; Genetic; Genetic Polymorphism; Genomics; Glutamine; Goals; Granulomatous; Greater sac of peritoneum; Health; Human; Hydrolysis; Immune system; Immunity; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Response; Inheritance Patterns; Injection of therapeutic agent; Injury; Insecta; Intervention; Intra-Articular Injections; Joints; K-Series Research Career Programs; Knock-in Mouse; Lead; Leprosy; Leucine-Rich Repeat; Life; Link; Liver; Malt Grain; Mediating; Medicine; Mentors; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; N-terminal; Natural Immunity; Nucleotides; Organ; Participant; Pathogenesis; Pathology; Pattern; Peptide Sequence Determination; Peptidoglycan; Peritoneal; Peritonitis; Physiological; Play; Positioning Attribute; Pre-Clinical Model; Process; Proteins; Regulatory Element; Research; Role; Sarcoidosis; Scientist; Serum; Shapes; Signal Transduction; Skin; Solutions; Staging; Structure; Syndrome; Testing; Tissues; Toll-Like Receptor 2; Toll-like receptors; Training; Training Programs; Translating; Veterans; Wild Type Mouse; Work; arm; atopy; body system; cytokine; disease phenotype; experience; gain of function; genetic information; graft vs host disease; human disease; in vitro testing; in vivo; insight; interest; macrophage; member; microbial; pathogen; protein function; receptor; receptor function; response; skills

DESCRIPTION (provided by applicant): This is a Career Development Award-2 (CDA-2) application by Jae Dugan, Ph.D., mentored by Michael Davey, M.D., Ph.D. and Daniel Carr, Ph.D. The PI developed expertise in molecular bacteriology during his thesis work investigating mechanisms of antibiotic resistance in Chlamydia. His current research interests involve understanding how the innate immune system contributes to human disease. Toll-like receptors (TLR) and NOD-like receptors (NLR) function as "sensing" proteins to activate the innate immune system. This application focuses on one member of the NLR family called nucleotide oligomerization domain 2 (NOD2). Mutations in NOD2 are linked to Crohn's disease, sarcoidosis, asthma, atopy, peritonitis, graft- versus-host disease, leprosy and Blau syndrome (chronic granulomatous inflammation of the skin, eyes, and joints). One specific domain within the NOD2 protein is the nucleotide oligomerization domain (NOD), also referred to as the nucleotide binding domain (NBD). Mutations found in Blau syndrome cluster in the NOD domain (the most frequent being a glutamine at position 334 instead of an arginine, indicated as R334Q). Studies of the NOD domain in other NLR family members have shown this domain to have an ATP binding site, leading to hydrolysis of ATP, followed by self oligomerization. Oligomerization is thought to be critical for subsequent downstream events. Blau syndrome represents a unique opportunity to investigate how NOD2 can contribute to inflammatory disease in multiple organs. The work proposed here will test the following hypotheses: 1. The R334Q-NOD2 mutation causes increased ATP binding, hydrolysis and oligomerization of NOD2 compared to wild type protein 2. Macrophages from mice expressing one copy of the equivalent of the human mutation will have increased cytokine release, intracelular signalling and autophagy formation compared to wild type mice and 3. Mutant mice will show signs of spontaneous inflammation, or enhanced inflammatory responses after exposure to known activators of NOD2. These hypotheses will be tested in 3 aims. In aim 1 the PI will investigate the impact of the R334Q mutation by studying human NOD2 expressed in and purified from insect cells. ATP binding and hydrolysis and oligomerization of NOD2 will be tested using wild type and R334Q-NOD2. Aims 2 and 3 will employ a knock-in mouse created by the PI and described in the work accomplished section where one copy of NOD2 carries the murine equivalent (R314Q) of the human R334Q mutation. Macrophages prepared from bone marrow and peritoneal cavity will be studied for cytokine response, intracellular signalling and autophagosome formation in response to muramyl dipeptide (MDP) a known activator of NOD2 (aim 2). Studies of synergy will also be performed with MDP and TLR agonists. Mutant mice will be carefully studied for signs of spontaneous inflamamtion and cytokine responses will be followed in sera following iv injection of MDP, MDP and TLR agonists, TLR agonists, and liver bacteria (aim 3). These studies have the potential to provide new and important insights into the mechanism by which NOD2 contributes to chronic inflammation. These insights should lead to rational, targeted interventions. The aims of this application also provide for a broad training experience for Dr. Dugan, greatly expanding his set of skills as he transitions to independence.

描述（由申请人提供）： 这是 Jae Dugan 博士的职业发展奖 2 (CDA-2) 申请，由 Michael Davey 医学博士指导。和丹尼尔·卡尔博士PI 在研究衣原体抗生素耐药机制的论文工作中发展了分子细菌学方面的专业知识。他目前的研究兴趣包括了解先天免疫系统如何导致人类疾病。 Toll 样受体 (TLR) 和 NOD 样受体 (NLR) 作为“传感”蛋白来激活先天免疫系统。本申请重点关注 NLR 家族的一个成员，称为核苷酸寡聚化结构域 2 (NOD2)。 NOD2 突变与克罗恩病、结节病、哮喘、特应性、腹膜炎、移植物抗宿主病、麻风病和布劳综合征（皮肤、眼睛和关节的慢性肉芽肿性炎症）有关。 NOD2 蛋白内的一个特定结构域是核苷酸寡聚化结构域 (NOD)，也称为核苷酸结合结构域 (NBD)。在 NOD 结构域中的 Blau 综合征簇中发现的突变（最常见的是位置 334 处的谷氨酰胺而不是精氨酸，表示为 R334Q）。对其他 NLR 家族成员的 NOD 结构域的研究表明，该结构域具有 ATP 结合位点，导致 ATP 水解，然后发生自身寡聚化。低聚被认为对于后续下游事件至关重要。 Blau 综合征为研究 NOD2 如何导致多个器官的炎症性疾病提供了独特的机会。 这里提出的工作将测试以下假设： 1. 与野生型蛋白相比，R334Q-NOD2 突变会导致 NOD2 的 ATP 结合、水解和寡聚增加 2. 表达一份相当于人类突变的小鼠的巨噬细胞会增加与野生型小鼠相比，细胞因子释放、细胞内信号传导和自噬形成以及3。突变型小鼠在暴露于已知的NOD2激活剂后将表现出自发炎症的迹象或增强的炎症反应。这些假设将在 3 个目标中得到检验。在目标 1 中，PI 将通过研究昆虫细胞中表达和纯化的人类 NOD2 来研究 R334Q 突变的影响。将使用野生型和 R334Q-NOD2 测试 NOD2 的 ATP 结合以及水解和寡聚化。目标 2 和 3 将使用 PI 创建的敲入小鼠，并在已完成的工作部分中进行描述，其中一个 NOD2 拷贝携带人类 R334Q 突变的小鼠等效物 (R314Q)。将研究从骨髓和腹膜腔制备的巨噬细胞对胞壁酰二肽 (MDP)（一种已知的 NOD2 激活剂）的细胞因子反应、细胞内信号传导和自噬体形成（目标 2）。还将与 MDP 和 TLR 激动剂进行协同作用研究。将仔细研究突变小鼠的自发炎症迹象，并在静脉注射 MDP、MDP 和 TLR 激动剂、TLR 激动剂和肝细菌后跟踪血清中的细胞因子反应（目标 3）。这些研究有可能为 NOD2 导致慢性炎症的机制提供新的重要见解。这些见解应该导致理性、有针对性的干预措施。此应用程序的目的还为杜根博士提供广泛的培训经验，在他向独立过渡时极大地扩展了他的技能。