REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

描述：这是对竞争续订提案的修订，以 大鼠呼吸爆发酶激活机制的研究 吞噬细胞。申请者是第一个细胞的开发者之一- 一种测定NADPH氧化酶活性的游离测定法 这一体系导致了许多成分的分子鉴定 到目前为止的氧化物酶。最近几年，认识到 最初的无细胞激活方案不需要ATP或蛋白质 通过磷酸化，申请人开发了一种新的无细胞激活 磷脂酸、甘油二酯和三磷酸腺苷联合使用的方案 中性粒细胞胞浆和细胞膜。根据申请人的学习情况 磷脂酶D的激活和磷脂酸的产生(PA) 在NADPH氧化酶的激活过程中，申请人形成了 目前PA plus对酶的无细胞激活方案 二酰甘油(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，在该系统中，蛋白激酶抑制剂对其中任何一个都没有影响 无细胞激活或活性，NADPH氧化酶活性在 无细胞PA/DG体系被蛋白质包被抑制75% 激酶抑制剂，如星形孢子素和H-7。三磷酸腺苷增强 体系的活性，而非水解性的ATP类似物不能， 但GTP-g-S的加入也增强了系统的活性， 但没有ATP加GTP-g-S那么多。申请者已经能够 表明该系统导致p47Phox和p47Phox的磷酸化。 P47的磷酸化先于氧化酶的活性。P47的身份 作为该体系中的一种磷酸化蛋白被确认 通过对一名P47Phox患者的胞浆进行实验 缺乏，其中没有检测到p47磷酸化，并且在 外源重组P47存在时的相同样本，其中 万一它被磷酸化了。这些数据表单和其他数据表单加在一起 新的具体目标的基础。第一个是净化，克隆 并表征了PA激活的蛋白激酶的磷酸化 重组p47Phox，第二次测定生理活性 PA激活的蛋白激酶与NADPH氧化酶的相关性 激活。申请人已开发出一种筛查试验，用于检测 重组p47的PA活化蛋白激酶的纯化 在32P-g-ATP存在下被磷酸化。该产品的来源 蛋白底物是杆状病毒感染Sf9细胞的上清 或在细菌中以GST融合蛋白的形式表达，两种结构都是 由申请者与之合作的Tom Leto提供。跟随 通过标准技术与Dr。 Reidar Wallin拥有广泛的蛋白质纯化专业知识，Sequence 将在全蛋白、胰酶或氰基上获得 溴化物片段，并最终使用来自 用于筛选蛋白质表达文库的序列。磷酸多肽 将首先在PA激活的蛋白激酶上进行作图 使用重组p47Phox的无细胞系统，然后模式 在完好无损的细胞中进行了验证。然后，磷酸化位点(S)将 指导建议书的下一部分，以创建和测试站点导向 突变形式的p47Phox确定个体的需求 P47Phox中激活NADPH氧化酶的磷酸化位点 无细胞系统和在完整细胞中。诱变的p47Phox 构建物将在Sf9昆虫细胞中表达并纯化用于 在无手机系统中使用。选定的突变cDNA将是 被整合到哺乳动物载体中，用于转染p47Phox- 缺陷细胞系，特别是在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转化的淋巴细胞的10倍 系统。另一个目标是完全重建超氧化物。 利用纯化的PA激活的蛋白激酶产生系统 纯化/重组NADPH氧化酶组分。纯化的细胞色素 B558将由Jesaitis博士和Quinn为这些研究捐赠。 最后，申请人将确定p47Phox的磷酸化是否 PA激活的蛋白激酶诱导NADPH的组装 通过p47Phox和p67Phox的SH3结构域。这将是 与Leto博士合作完成的一项实验中，GST-p22Phox 将融合蛋白应用于硝化纤维，并将其量 P47Phox与PA激活的磷酸化前后结合 蛋白激酶用抗p47Phox抗体测定。

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.