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Studies on the phagocyte-specific NADPH oxidase involving in the killing of micro organisms and cell damage and the mechanism of activation

吞噬细胞特异性NADPH氧化酶参与微生物杀伤和细胞损伤及其激活机制的研究

基本信息

批准号：
04454173
负责人：
TAKESHIGE Koichiro
金额：
$ 4.1万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454173/
关键词：
Neutrophils NADPH oxidase Active oxygens Superoxide Signal transduction 細胞障害スーパーオキシド

项目摘要

(1)Studies in a cell-free system (i)Because the alignments of the amino acid sequence of the large subunit of the cytochrome b558 with those of previously characterized flavoproteins revealed that the middle and C-terminal portions of the cytochrome are likely to be FAD-and NADPH-binding domains, respectively, cytochrome b558 appears to be a flavoprotein with an NADPH- binding site of the NADPH oxidase. (ii)We showed that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore we found that sodium dodecyl sulfate and arachidonic acid, activators of the oxidase in vitro, caused exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appeared to play a crucial role in the assembly of the oxidase components, because p47-SH3 bound to b … More oth p22phox ana p67phox but failed to interact with a mutant p22phox carrying Pro156Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a novel signal-transducing mechanism that normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins. (iii)Phorbol 12-myristate 13-acetate (PMA) induced a decrease in the phosphotyrosine phosphatase (PTPase) activity in human neutrophils. The decrease in the activity induced by PMA was blocked by the treatment of the cells with staurosporine, indicating that protein kinase C is involved in the decrease. The findings obtained suggest that conformational changes of the enzyme induced by PMA result in the decrease in PTPase activity.(2)Studies with intact or electropermeabilized neutrophils We showed by using electropermeabilized human neutrophils that cAMP inhibited the activation of the NADPH oxidase not only the at the site before the protein kinase C but also at another site after the kinase, that tyrosine phosphorylation is involved in the activation of the NADPH oxidase at a step before diacylglycerol formation by phospholipase C and phospholipase D may not be involived the signal pathway in permeabilized cells and that phosphatidic acid activates the oxidase at a site down stream of protein kinase C. Less

（1）在无细胞系统中的研究（i）由于细胞色素b558大亚基的氨基酸序列与先前表征的黄素蛋白的氨基酸序列的比对显示，细胞色素的中间和C末端部分可能分别是FAD和NADPH结合结构域，细胞色素b558似乎是具有NADPH氧化酶的NADPH结合位点的黄素蛋白。（ii）我们发现，p47 phox（p47-SH 3）的串联SH 3结构域的区域表达为谷胱甘肽S-转移酶融合抑制超氧化物的产生在无细胞系统中，表明参与激活的结构域。此外，我们发现，十二烷基硫酸钠和花生四烯酸，在体外的氧化酶的激活剂，引起暴露的p47-SH 3，这可能是被掩盖的C-末端区域的这种蛋白质在静息状态。p47-SH 3的暴露似乎在氧化酶组分的组装中起关键作用，因为p47-SH 3与B结合 ...更多信息 p22 phox和p67 phox均不能与突变型p22 phox相互作用，该突变型p22 phox在富含脯氨酸的区域携带Pro 156 Gln取代，该突变型p22 phox在慢性肉芽肿病患者中被发现。基于这些观察，我们提出了一种新的信号转导机制，即通常不可接近的SH 3结构域在激活后与其靶蛋白相互作用。（iii）佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）诱导人类中性粒细胞中磷酸酪氨酸磷酸酶（PTPase）活性降低。PMA诱导的活性降低被星形孢菌素处理的细胞所阻断，表明蛋白激酶C参与了这种降低。结果表明，PMA诱导的酶构象变化导致PTB活性下降。（2）用完整的或电透性的中性粒细胞进行的研究我们用电透性的人中性粒细胞表明，cAMP不仅在蛋白激酶C之前的位点抑制NADPH氧化酶的活化，而且在激酶之后的另一个位点也抑制NADPH氧化酶的活化，在磷脂酶C和磷脂酶D形成二酰甘油之前的一个步骤中，酪氨酸磷酸化参与了NADPH氧化酶的激活，但可能不参与其中。磷脂酸激活蛋白激酶C下游位点氧化酶。少