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

(1)由于细胞色素b558的大亚基氨基酸序列与先前表征的黄蛋白的氨基酸序列的对比表明，细胞色素的中间和c端部分可能分别是fad和NADPH结合域，因此细胞色素b558似乎是具有NADPH氧化酶NADPH结合位点的黄蛋白。（ii）我们发现p47phox串联SH3结构域的区域（p47-SH3）表达为谷胱甘肽s转移酶融合，在无细胞系统中抑制超氧化物的产生，表明该结构域参与了激活。此外，我们发现十二烷基硫酸钠和花生四烯酸是体外氧化酶的激活剂，可引起p47-SH3的暴露，而p47-SH3在静息状态下可能被该蛋白的c端区域所掩盖。p47-SH3的发现似乎在氧化酶组分的组装中起着至关重要的作用，因为p47-SH3与p22phox和p67phox结合，但不能与p22phox突变体相互作用，p22phox突变体在富含脯氨酸的区域携带Pro156Gln替代，这在慢性肉芽肿病患者中被发现。基于观察结果，我们提出了一种新的信号转导机制，即正常情况下无法进入的SH3结构域在激活后暴露并与其靶蛋白相互作用。（iii）Phorbol 12-肉豆蔻酸酯13-乙酸酯（PMA）诱导人中性粒细胞磷酸化酪氨酸磷酸酶（PTPase）活性降低。经staurosporine处理后，PMA诱导的活性下降被阻断，表明蛋白激酶C参与了活性下降。结果表明，PMA诱导的PTPase构象变化导致PTPase活性降低。(2)完整中性粒细胞或电渗透中性粒细胞的研究我们发现，通过电渗透人中性粒细胞，cAMP不仅在蛋白激酶C之前的位点抑制NADPH氧化酶的激活，而且在激酶C之后的另一个位点也抑制NADPH氧化酶的激活。在磷脂酶C和磷脂酶D形成二酰基甘油之前，酪氨酸磷酸化参与了NADPH氧化酶的激活，而磷脂酶D可能不参与渗透细胞的信号通路，磷脂酸激活了蛋白激酶C下游的氧化酶

(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