Redox regulation of phagocyte NOX2 in inflammation and aging

吞噬细胞NOX2在炎症和衰老中的氧化还原调节

• 批准号: 7931541
• 负责人: ROBERT A CLARK
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931541
• 关键词: ABL1 gene; Age; Aging; Aging-Related Process; Autoimmunity; Binding; Biochemical; Biological Assay; Biological Process; Biology of Aging; Calcium; Cations; Cell Aging; Cell membrane; Cells; Cellular biology; Characteristics; Chronic; Chronic Granulomatous Disease; Complex; Data; Deterioration; Disease; Drug or chemical Tissue Distribution; Elderly; Elements; Enzymes; Event; Family; Feedback; Functional disorder; GTP-Binding Proteins; Gene Expression; Gene Family; Generations; Genes; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Homologous Gene; Host Defense; Human; Hydrogen Peroxide; Immune; In Vitro; Individual; Infection; Inflammation; Inflammatory; Injury; Invaded; Laboratories; Lead; Leukocytes; Linear Models; Malignant Neoplasms; Measures; Mediating; Membrane; Microbe; Molecular; Molecular Biology; Morbidity - disease rate; Myelogenous; NADPH Oxidase; NADPH Oxidase 1; Natural Immunity; Oxidases; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Pathway interactions; Patients; Pattern; Performance; Phagocytes; Phenotype; Phosphorylation; Phosphotransferases; Play; Predisposition; Protein Chemistry; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Publications; Reactive Oxygen Species; Reagent; Recurrence; Regulation; Regulatory Pathway; Research; Role; SRC gene; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Source; Stimulus; Superoxides; System; TRPC3 ion channel; Techniques; Testing; Tissues; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; Work; abstracting; age related; antimicrobial; base; c-abl Proto-Oncogenes; cytochrome b558; design; feeding; frailty; human CYBA protein; human subject; in vivo; inflammatory marker; innovation; interest; killings; member; microbicide; microorganism; monocyte; mortality; mutant; neutrophil; neutrophil cytosol factor 67K; novel; programs; research study; response; rho GTP-Binding Proteins; sulfated glycoprotein 2; superoxide-generating NADPH oxidase; transcription factor; voltage; young adult

DESCRIPTION (provided by applicant): Project Summary / Abstract Neutrophils comprise the first line of host defense, killing invading microbes by generating reactive oxygen species (ROS) through an activatable NADPH oxidase system. This respiratory burst oxidase is genetically absent in patients with chronic granulomatous disease, resulting in recurring severe infections with high morbidity and mortality. The phagocyte oxidase features membrane-bound catalytic components (gp91phox/p22phox) and cytosolic co-factors (p47phox, p67phox, Rac1/2) that undergo stimulus-dependent translocation during assembly of the active NADPH oxidase. Recently, it has been found that gp91phox is actually the founder of the 7-member NOX/DUOX gene family, each homologue having its characteristic pattern of tissue expression and functional role. We have recently demonstrated a novel feed-forward regulation of NOX5 by hydrogen peroxide (H2O2) acting through a signaling pathway involving calcium and the non-receptor tyrosine kinase c-Abl. Especially relevant to the current proposal are our preliminary data suggesting that a related pathway exists for the phagocyte NADPH oxidase (NOX2) and that this pathway is dysregulated with aging, a biological process associated with ROS and oxidative stress. Our main objectives are to characterize the regulation of NOX2 in human phagocytic cells by H2O2 and to assess the relevance of such regulation to chronic inflammation and aging. Our hypothesis is that H2O2 is a common regulator of the NOX enzymes through specific redox-mediated signaling pathways and that this mechanism is dysregulated during aging. Three specific aims will guide the work. Aim 1 is to dissect the proximal elements of this signal transduction pathway as a means of defining the early targets of H2O2, focusing on the H2O2-activated Ca2+ channels involved, the mechanisms of channel regulation, and the signaling intermediates, especially protein tyrosine kinases (PTKs), that are directly regulated by Ca2+ influx. Aim 2 is to investigate the intermediates that lead from H2O2-activated PTKs (c-Abl, c-Src) to assembly and activation of NOX2, considering the role of specific PKC isotypes, the mechanism of activation of Rac GTPase, and the direct tyrosine phosphorylation of NOX2. Aim 3 is to examine the status and role of H2O2-NOX2 regulation in human phagocytic leukocytes as a function of age, including both cross-sectional and longitudinal assessment of NOX2-mediated superoxide generation, correlation with biochemical and functional markers of the aging phenotype, and mechanistic exploration of the signaling pathways defined in Aims 1 and 2. The studies proposed are innovative because they capitalize on our novel observations on redox regulation of NOX, and take advantage of key reagents, constructs, and cells, as well as extensive preliminary data and the broad-based expertise of our research team. The experiments will use state-of-the-art techniques in cell biology, protein chemistry, and molecular biology. The scientific significance of our proposal is that it has the potential for establishing broadly applicable precedents for the cell biology of ROS generation, host defenses, and cell signaling, as well as aging-associated ROS dysregulation. PUBLIC HEALTH RELEVANCE: Project Narrative The veterans health relevance of this proposal is that the results are likely to have significant implications for a variety of diseases that are highly prevalent in veterans, as well as in the general population. NOX2- derived toxic reactive oxygen species are presently known to be involved in the pathogenesis of a number of disorders, including among others atherosclerosis, myocardial infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), and ischemic stroke. Moreover, there is convincing evidence that NOX-derived toxic oxygen products are involved in the aging process. Since this project will address regulation of the production of the damaging forms of oxygen that mediate such disorders, there is considerable potential for improved understanding, as well as the identification of new targets for therapeutic intervention for the benefit of patients with these diseases, as well as for the amelioration of certain aspects of the aging process.

描述（由申请人提供）： 中性粒细胞是宿主防御的第一道防线，通过可激活的NADPH氧化酶系统产生活性氧（ROS）来杀死入侵的微生物。这种呼吸爆发氧化酶在慢性肉芽肿病患者中遗传缺失，导致复发性严重感染，发病率和死亡率高。吞噬细胞氧化酶的特点是膜结合的催化成分（gp 91 phox/p22 phox）和胞质辅因子（p47 phox，p67 phox，Rac 1/2），在活性NADPH氧化酶组装过程中经历刺激依赖性易位。最近，人们发现gp 91 phox实际上是NOX/DUOX基因家族7个成员的创始人，每个同源物都有其特征性的组织表达模式和功能作用。我们最近证明了过氧化氢（H2 O2）通过涉及钙和非受体酪氨酸激酶c-Abl的信号通路作用于NOX 5的一种新的前馈调节。与目前的提议特别相关的是我们的初步数据，表明吞噬细胞NADPH氧化酶（NOX 2）存在相关通路，并且该通路随着衰老而失调，一种与ROS和氧化应激相关的生物过程。 我们的主要目标是表征H2 O2对人类吞噬细胞中NOX 2的调节，并评估这种调节与慢性炎症和衰老的相关性。我们的假设是，H2 O2是一种常见的调节剂的NOX酶通过特定的氧化还原介导的信号通路，这种机制是失调，在老化过程中。三个具体目标将指导这项工作。目的1是剖析这一信号转导途径的近端元件，作为确定H2 O2早期靶点的手段，重点是H2 O2激活的Ca 2+通道，通道调节机制，以及信号中间体，特别是蛋白酪氨酸激酶（PTKs），直接受Ca 2+内流的调节。目的二是研究H2 O2激活的PTKs（c-Abl、c-Src）组装和激活NOX 2的中间产物，包括PKC的作用、Rac GT3的激活机制以及NOX 2的直接酪氨酸磷酸化。目的3是研究H2 O2-NOX 2调节在人类吞噬白细胞中的地位和作用，作为年龄的函数，包括NOX 2介导的超氧化物生成的横截面和纵向评估，与衰老表型的生化和功能标志物的相关性，以及目的1和2中定义的信号传导途径的机制探索。 提出的研究是创新的，因为它们利用了我们对NOX氧化还原调节的新观察，并利用了关键试剂，构建体和细胞，以及广泛的初步数据和我们研究团队的广泛专业知识。这些实验将使用细胞生物学、蛋白质化学和分子生物学的最先进技术。我们的建议的科学意义在于，它有可能为ROS生成，宿主防御和细胞信号传导以及衰老相关的ROS失调的细胞生物学建立广泛适用的先例。 公共卫生关系： 该提案与退伍军人健康的相关性在于，其结果可能对退伍军人以及普通人群中高度流行的各种疾病产生重大影响。目前已知NOX 2衍生的毒性活性氧物质参与许多疾病的发病机制，包括动脉粥样硬化、心肌梗塞、阿尔茨海默病、帕金森病、肌萎缩侧索硬化（Lou Gehrig病）和缺血性中风等。此外，有令人信服的证据表明，NOX衍生的有毒氧产物参与了衰老过程。由于该项目将解决调节介导这些疾病的有害形式的氧气的产生，因此有相当大的潜力来提高理解，以及确定新的治疗干预目标，以使这些疾病的患者受益，以及改善衰老过程的某些方面。