REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

血液中性粒细胞氧代谢的调节

• 批准号: 6124188
• 负责人: LINDA C. McPHAIL
• 金额: $ 29.29万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2001-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124188
• 关键词: NAD(P)H dehydrogenase; Sf9 cell line; cell free system; chimeric proteins; clinical research; complementary DNA; cytochrome b; enzyme activity; human subject; leukocyte oxidative burst; molecular cloning; neutrophil; phosphopeptides; phosphorylation; protein kinase; protein purification; protein sequence; recombinant proteins; respiratory oxygen; site directed mutagenesis; superoxides; transfection /expression vector

DESCRIPTION: This is a revision of a competing renewal proposal to study mechanisms of activation of the respiratory burst oxidase of phagocytes. The Applicant was one of the developers of the first cell- free assay system for activation of NADPH oxidase activity, an assay system that led to much of the molecular identification of components of the oxidase to date. In more recent years, recognizing that the original cell-free activation scheme did not require ATP or protein phosphorylation, the Applicant developed a novel cell-free activation scheme involving phosphatidic acid, diacylglycerol and ATP in conjunction with neutrophil cytosol and membranes. Based on the Applicant's studies of phospholipase D activation and phosphatidic acid production (PA) during the activation of the NADPH oxidase, the applicant developed the current cell-free activation scheme of the oxidase by PA plus diacylglycerol (DG), indicating that the products of phospholipase D activity are able to activate the oxidase. In contrast to the SDS/DG system, in which protein kinase inhibitors had no effect on either cell-free activation or activity, NADPH oxidase activity activated in the cell-free PA/DG system was inhibited by 75% by inclusion of protein kinase inhibitors such as staurosporine and H-7. ATP enhances the activity of the system, whereas non-hydrolyzable ATP analogs do not, but the inclusion of GTP-g-S also enhances the activity of the system, but not as much as ATP plus GTP-g-S. The Applicant has been able to show that the system results in phosphorylation of p47phox and that p47 phosphorylation precedes oxidase activity. The identity of p47 as one of the phosphorylated proteins in this system was confirmed by performing the experiment with cytosol from a patient with p47 phox deficiency, in which no p47 phosphorylation was detected, and in the same sample when exogenous recombinant p47 was present, in which case it was phosphorylated. Taken together, these and other data form the basis of the new specific aims. The first is to purify, clone and characterize the PA-activated protein kinase that phosphorylates recombinant p47phox, and the second to determine the physiological relevance of the PA-activated protein kinase for NADPH oxidase activation. The Applicant has developed a screening assay for purification of the PA-activated protein kinase in which recombinant p47 is phosphorylated in the presence of 32P-g-ATP. The source of the protein substrate is the supernatant of baculovirus infected Sf9 cells or expressed in bacteria as a GST fusion protein, both constructs being provided by Tom Leto, with whom the applicant collaborates. Following purification by standard techniques with the collaboration of Dr. Reidar Wallin who has extensive protein purification expertise, sequence will be obtained on either the whole protein or tryptic or cyanogen bromide fragments, and eventually cloned using antibodies derived from sequence to screen protein-expressing libraries. The phosphopeptide mapping will be performed first on PA-activated protein kinase in the cell free system using recombinant p47phox, and then the patterns verified in the intact cell. The phosphorylation site(s) will then direct the next part of the proposal, to create and test site-directed mutagenized forms of p47phox to determine the requirement of individual phosphorylation sites in p47phox for activation of NADPH oxidase the cell-free system and in intact cells. The mutagenized p47phox constructs will be expressed in Sf9 insect cells and purified for use in the cell-free system. Selected mutant CDNAS will be incorporated into mammalian vectors for transfection into p47phox- deficient cell lines, specifically in a K562 cell line model missing only p47 and p67, developed by Dr. Tom Leto, a cell line model that generates 10-fold more superoxide than the EBV-transformed lymphocyte systems. An additional aim is to fully reconstitute the superoxide generating system using purified PA-activated protein kinase with purified/recombinant NADPH oxidase components. The purified cytochrome b558 will be donated by Dr. Jesaitis and Quinn for these studies. Lastly, the Applicant will determine whether phosphorylation of p47phox by the PA-activated protein kinase induces assembly of NADPH oxidase via the SH3 domains of p47phox and p67phox. This will be done in collaboration with Dr. Leto, in an assay in which GST-p22phox fusion proteins are applied to nitrocellulose, and the amount of p47phox bound before and after phosphorylation with PA-activated protein kinase determined using an anti-p47phox antibody.

描述：这是对竞争性更新提案的修订 呼吸爆发氧化酶激活机制的研究 吞噬细胞。 申请人是第一个细胞的开发商之一 用于激活 NADPH 氧化酶活性的免费检测系统，一种检测 导致大部分成分分子鉴定的系统 迄今为止的氧化酶。 近年来，人们认识到 原始的无细胞激活方案不需要ATP或蛋白质 磷酸化，申请人开发了一种新型的无细胞激活 涉及磷脂酸、二酰甘油和ATP联合的方案 具有中性粒细胞胞质和细胞膜。 基于申请人的研究 磷脂酶 D 激活和磷脂酸生成 (PA) 在NADPH氧化酶的激活过程中，申请人开发了 目前PA plus无细胞激活氧化酶的方案 二酰基甘油(DG)，表明磷脂酶D的产物 活性能够激活氧化酶。 与 SDS/DG 相比 系统，其中蛋白激酶抑制剂对两者都没有影响 无细胞激活或活性，NADPH氧化酶活性在 通过包含蛋白质，无细胞 PA/DG 系统被抑制 75% 激酶抑制剂，例如十字孢菌素和H-7。 ATP 增强 系统的活性，而不可水解的 ATP 类似物则不会， 但GTP-g-S的加入也增强了系统的活性， 但不如 ATP 加 GTP-g-S 那么多。 申请人已经能够 表明该系统导致 p47phox 磷酸化和 p47 磷酸化先于氧化酶活性。 p47的身份 作为该系统中的磷酸化蛋白质之一被证实 通过使用 p47 p47 phox 患者的细胞质进行实验 缺陷，其中未检测到 p47 磷酸化，并且 存在外源重组 p47 时的同一样品，其中 如果它被磷酸化。综合起来，这些数据和其他数据形成 新的具体目标的基础。首先是纯化、克隆 并表征磷酸化的 PA 激活蛋白激酶 重组p47phox，第二次测定生理学 PA 激活蛋白激酶与 NADPH 氧化酶的相关性 激活。 申请人开发了一种筛选测定法 PA 激活蛋白激酶的纯化，其中重组 p47 在 32P-g-ATP 存在下被磷酸化。的来源 蛋白质底物是杆状病毒感染的Sf9细胞的上清液 或在细菌中表达为 GST 融合蛋白，两种构建体都是 由与申请人合作的 Tom Leto 提供。下列的 在 Dr. 的合作下通过标准技术进行纯化 Reidar Wallin 拥有丰富的蛋白质纯化、序列专业知识 将从整个蛋白质或胰蛋白酶或氰中获得 溴化物片段，并最终使用衍生自的抗体进行克隆 筛选蛋白质表达文库的序列。磷酸肽 首先将在 PA 激活蛋白激酶上进行作图 使用重组 p47phox 的无细胞系统，然后得到模式 在完整的细胞中得到验证。然后磷酸化位点将 指导提案的下一部分，创建和测试现场定向 p47phox 的诱变形式以确定个体的需求 p47phox 中用于激活 NADPH 氧化酶的磷酸化位点 无细胞系统和完整细胞中。 诱变的 p47phox 构建体将在 Sf9 昆虫细胞中表达并纯化 在无细胞系统中使用。选定的突变体 CDNAS 将是 掺入哺乳动物载体以转染 p47phox- 缺陷细胞系，特别是 K562 细胞系模型缺失 只有 p47 和 p67，由 Tom Leto 博士开发的细胞系模型 产生的超氧化物是 EBV 转化淋巴细胞的 10 倍 系统。 另一个目标是完全重建超氧化物 使用纯化的 PA 激活蛋白激酶生成系统 纯化/重组 NADPH 氧化酶成分。纯化的细胞色素 b558 将由 Jesaitis 博士和 Quinn 捐赠用于这些研究。 最后，申请人将确定p47phox是否磷酸化 PA 激活蛋白激酶诱导 NADPH 组装 氧化酶通过 p47phox 和 p67phox 的 SH3 结构域。这将是 与 Leto 博士合作进行的一项检测中，GST-p22phox 将融合蛋白应用于硝化纤维素，并且用量 p47phox 在 PA 激活磷酸化之前和之后结合 使用抗 p47phox 抗体测定蛋白激酶。