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: 8142539
• 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/8142539
• 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; 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)： 这是由迈克尔·戴维医学博士和丹尼尔·卡尔博士指导的Jae Dugan博士的职业发展奖-2(CDA-2)申请。PI在他的论文工作中发展了分子细菌学方面的专业知识，研究衣原体的抗生素耐药机制。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”蛋白激活先天免疫系统。该应用集中在NLR家族中的一个成员，称为核苷酸寡聚化结构域2(NOD2)。NOD2基因突变与克罗恩病、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蛋白中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BLAU综合征中发现了NOD结构域的突变(最常见的是第334位的谷氨酰胺，而不是精氨酸，表示为R334Q)。对NLR家族其他成员的Nod结构域的研究表明，该结构域具有一个ATP结合部位，导致ATP的水解，随后发生自我寡聚。寡聚化被认为是后续下游事件的关键。BLAU综合征是一个独特的机会，可以研究NOD2如何导致多个器官的炎症性疾病。这项工作将检验以下假设：1.R334Q-NOD2突变导致与野生型蛋白2相比，NOD2的ATP结合、水解性和寡聚化增加。2.表达人类突变等价物的小鼠的巨噬细胞与野生型小鼠相比，细胞因子释放、细胞内信号传递和自噬形成增加。3.突变小鼠在暴露于已知的NOD2激活剂后，将显示出自发性炎症或炎症反应增强的迹象。这些假设将在三个目标中得到检验。在目标1中，PI将通过研究人类NOD2在昆虫细胞中的表达和纯化来研究R334Q突变的影响。将使用野生型和R334Q-NOD2来测试ATP结合以及NOD2的水解性和寡聚化。AIMS 2和3将使用PI创建的敲入小鼠，并在工作完成部分进行描述，其中NOD2的一个副本携带人类R334Q突变的小鼠等价物(R314Q)。从骨髓和腹膜腔制备的巨噬细胞将研究细胞因子反应、细胞内信号和自噬小体的形成，以响应已知的NOD2激活剂胞壁二肽(MDP)(目标2)。协同作用的研究也将与MDP和TLR激动剂进行。将仔细研究突变小鼠的自发性炎症迹象，并跟踪静脉注射MDP、MDP和TLR激动剂、TLR激动剂和肝脏细菌后血清中的细胞因子反应(目标3)。这些研究有可能为NOD2促进慢性炎症的机制提供新的和重要的见解。这些洞察力应该会导致理性的、有针对性的干预。这款应用程序的目标还为杜根博士提供了广泛的培训经验，在他过渡到独立的过程中极大地扩展了他的技能集。 公共卫生相关性： NOD2与许多慢性炎症性疾病有关，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。我们对先天免疫介导的疾病的理解还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的炎症的发展，将对退伍军人的健康产生广泛的影响。