REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

描述：这是一个竞争性更新提案的修订版， 研究呼吸爆发氧化酶的激活机制， 吞噬细胞 申请人是第一个细胞的开发者之一- 用于激活NADPH氧化酶活性的自由测定系统， 这个系统导致了许多成分的分子鉴定 最新的氧化酶。 近年来，认识到 最初的无细胞激活方案不需要ATP或蛋白质 为了抑制磷酸化，申请人开发了一种新的无细胞活化方法， 涉及磷脂酸、二酰甘油和ATP联合的方案 中性粒细胞胞浆和细胞膜。 根据申请人的研究 磷脂酶D活化和磷脂酸产生（PA） 在NADPH氧化酶的活化过程中，申请人开发了 目前的无细胞激活方案的氧化酶PA加 甘油二酯（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 无细胞PA/DG系统被蛋白包合物抑制75 激酶抑制剂如星形孢菌素和H-7。 ATP增强了 活性的系统，而不可水解的ATP类似物没有， 但是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。 申请人已能够 显示该系统导致p47 phox的磷酸化， 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， 通过用来自p47 phox患者的胞质溶胶进行实验 缺乏，其中没有检测到p47磷酸化，并且在 当存在外源重组p47时，使用相同的样品，其中 是磷酸化的综合起来，这些数据和其他数据 新的具体目标的基础。首先是提纯、克隆 并表征磷酸化PA活化的蛋白激酶， 重组p47 phox，第二个是确定生理 PA活化蛋白激酶与NADPH氧化酶的相关性 activation. 申请人已经开发了一种筛选测定法， 纯化PA活化蛋白激酶，其中重组p47 在32 P-g-ATP存在下磷酸化。的来源 蛋白质底物是杆状病毒感染的Sf 9细胞的上清液 或在细菌中表达为GST融合蛋白，两种构建体均 由汤姆·莱托提供，申请人与他合作。以下 通过标准技术与Dr. Reidar Wallin拥有丰富的蛋白质纯化专业知识， 将在整个蛋白质或胰蛋白酶或氰上获得 溴片段，并最终克隆使用抗体来源于 序列来筛选蛋白表达文库。述磷酸肽 将首先对PA激活的蛋白激酶进行定位， 利用重组p47 phox的无细胞系统， 在完整的细胞中验证。磷酸化位点将 指导下一部分的建议，建立和测试网站导向 突变形式的p47 phox，以确定个人的需求 p47 phox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。 突变的p47 phox 构建体将在Sf 9昆虫细胞中表达，并纯化用于纯化。 在无细胞系统中使用。选择的突变体CDNAS将是 用于转染入p47 phox- 缺陷细胞系，特别是在缺失的K562细胞系模型中 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EB病毒转化的淋巴细胞的10倍 系统. 另一个目的是完全重建超氧化物 使用纯化的PA活化蛋白激酶的产生系统， 纯化的/重组的NADPH氧化酶组分。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p47 phox的磷酸化是否 通过PA活化的蛋白激酶诱导NADPH的组装 通过p47 phox和p67 phox的SH 3结构域对氧化酶进行修饰。这将是 在与Leto博士合作的一项试验中，GST-p22 phox 将融合蛋白应用于硝化纤维素， p47 phox在磷酸化前后与PA活化的 使用抗p47 phox抗体测定蛋白激酶。

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.