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: 8261858
• 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/8261858
• 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; graft vs host disease; human disease; in vitro testing; in vivo; insight; interest; macrophage; member; microbial; pathogen; protein function; public health relevance; 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. PUBLIC HEALTH RELEVANCE: NOD2 has been implicated in many chronic inflammatory conditions including Crohn's Disease, sarcoidosis, atopy, peritonitis and graft-versus-host disease. These diseases are important in veteran's health. Our understanding of diseases mediated by innate immunity is just in the early stages of development. Understanding how NOD2 controls the development of inflammation leading to tissue injury and pathology will have broad implications for the health of veterans.

描述（由申请人提供）： 这是Jae Dugan，Ph.D。的职业发展奖2（CDA-2）申请，由M.D. M.D.，Ph.D。指导。和丹尼尔·卡尔（Daniel Carr）博士PI在他的论文工作中研究了衣原体的抗生素耐药性机制。他目前的研究兴趣涉及了解先天免疫系统如何促进人类疾病。 Toll样受体（TLR）和NOD样受体（NLR）充当“传感”蛋白，以激活先天免疫系统。该应用集中在一个名为核苷酸寡聚结构域2（NOD2）的NLR家族的一个成员上。 NOD2中的突变与克罗恩病，结节疾病，哮喘，特应性，腹膜炎，移植物与宿主病，麻风病和BLAU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结构域（NOD），也称为核苷酸结合结构域（NBD）。在NOD结构域中BLAU综合征簇中发现的突变（最常见的是位置334的谷氨酰胺，而不是精氨酸，称为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，然后进行自寡聚。人们认为寡聚对于随后的下游事件至关重要。 BLAU综合征是研究NOD2如何为多个器官炎症性疾病做出贡献的独特机会。 这里提出的工作将测试以下假设：1。与野生型蛋白2相比，NOD2的R334Q-NOD2突变导致NOD2的ATP结合，水解和寡聚化增加。表达人类突变等效的一份副本的巨噬细胞将增加人类突变的副本，使胞质的信号和3次突变体形式和3次体内的群体相比，与3个散发群体相比。暴露于已知的NOD2激活剂后，自发炎症或增强的炎症反应。这些假设将以3个目标进行检验。在AIM 1中，PI将通过研究在昆虫细胞中表达并纯化的人NOD2来研究R334Q突变的影响。 NOD2的ATP结合和水解和寡聚化将使用野生型和R334Q-NOD2进行测试。 Aim 2和3将采用由PI创建的敲入小鼠，并在完成的部分中描述了一个NOD2的副本，其中一个副本带有人类R334Q突变的鼠等效（R314Q）。将研究由骨髓和腹膜腔制备的巨噬细胞，以响应已知的NOD2激活剂，以响应穆拉米基二肽（MDP），用于细胞因子反应，细胞内信号传导和自噬体形成（AIM 2）。 Synergy的研究也将使用MDP和TLR激动剂进行。将仔细研究突变小鼠的自发性症状迹象，在静脉注射MDP，MDP和TLR激动剂，TLR激动剂和肝细菌后，将在血清中遵循细胞因子反应（AIM 3）。这些研究有可能对NOD2有助于慢性炎症的机制提供新的重要见解。这些见解应导致理性，有针对性的干预措施。该应用程序的目的还为Dugan博士提供了广泛的培训经验，在过渡到独立性时，大大扩展了他的技能。 公共卫生相关性： NOD2与许多慢性炎症疾病有关，包括克罗恩病，结节病，特应性，腹膜炎和移植物抗宿主病。这些疾病对退伍军人的健康很重要。我们对由先天免疫介导的疾病的理解正处于发展的早期阶段。了解NOD2如何控制导致组织损伤和病理学的炎症发展将对退伍军人的健康产生广泛的影响。