De Leo F R, Ulman K V, Davis A R, Jutila K L, Quinn M T
Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59715, USA.
J Biol Chem. 1996 Jul 19;271(29):17013-20. doi: 10.1074/jbc.271.29.17013.
The human neutrophil NADPH oxidase is a multi-component complex composed of membrane-bound and cytosolic proteins. During activation, cytosolic proteins p47(phox), p67(phox), Rac2, and possibly p40(phox) translocate to the plasma membrane and associate with flavocytochrome b to form the active superoxide-generating system. To further investigate the role of p67(phox) in this complex assembly process, experiments were performed to identify possible regions of interaction between p67(phox) and other NADPH oxidase proteins. Using random sequence peptide phage-display library analysis of p67(phox), we identified a novel region in p47(phox) encompassing residues 323-332 and a previously identified SH3 binding domain encompassing p47(phox) residues 361-370 as p67(phox) binding sites. Synthetic peptides mimicking p47(phox) residues 323-332 inhibited the p47(phox)-p67(phox) binding interaction in an affinity binding assay; however, peptides mimicking flanking regions were inactive. Surprisingly, this same region of p47(phox) was found previously to represent a site of binding interaction for flavocytochrome b (DeLeo, F. R., Nauseef, W. M., Jesaitis, A. J., Burritt, J. B., Clark, R. A., and Quinn, M. T.(1995) J. Biol. Chem. 270, 26246-26251), and this observation was confirmed in the present report using two different in vitro assays that were not evaluated previously. Using affinity binding assays, we also found that p67(phox) and flavocytochrome b competed for binding to p47(phox)after activation, suggesting that prior to full NADPH oxidase assembly the 323-332 region of p47(phox) is associated with p67(phox) and at some point in the activation process is transferred to flavocytochrome b. Thus, taken together our data demonstrate that both p67(phox) and flavocytochrome b utilize a common binding site in p47(phox), presumably at distinct stages during the activation process, and this p47(phox) region plays a key role in regulating NADPH oxidase assembly.
人类中性粒细胞NADPH氧化酶是一种由膜结合蛋白和胞质蛋白组成的多组分复合物。在激活过程中，胞质蛋白p47（phox）、p67（phox）、Rac2以及可能的p40（phox）转位至质膜，并与黄素细胞色素b结合，形成活性超氧化物生成系统。为了进一步研究p67（phox）在该复合物组装过程中的作用，进行了实验以确定p67（phox）与其他NADPH氧化酶蛋白之间可能的相互作用区域。通过对p67（phox）进行随机序列肽噬菌体展示文库分析，我们在p47（phox）中鉴定出一个包含323 - 332位残基的新区域以及一个先前鉴定的包含p47（phox）361 - 370位残基的SH3结合域作为p67（phox）的结合位点。在亲和结合试验中，模拟p47（phox）323 - 332位残基的合成肽抑制了p47（phox） - p67（phox）的结合相互作用；然而，模拟侧翼区域的肽则无活性。令人惊讶的是，先前发现p47（phox）的同一区域代表黄素细胞色素b的结合相互作用位点（德利奥，F.R.，瑙西夫，W.M.，耶赛蒂斯，A.J.，伯里特，J.B.，克拉克，R.A.和奎因，M.T.（1995年）《生物化学杂志》270，26246 - 26251），并且本报告使用两种先前未评估的不同体外试验证实了这一观察结果。通过亲和结合试验，我们还发现激活后p67（phox）和黄素细胞色素b竞争与p47（phox）的结合，这表明在NADPH氧化酶完全组装之前，p47（phox）的323 - 332区域与p67（phox）相关联，并且在激活过程的某个时刻转移至黄素细胞色素b。因此，综合我们的数据表明，p67（phox）和黄素细胞色素b在p47（phox）中利用一个共同的结合位点，大概是在激活过程的不同阶段，并且这个p47（phox）区域在调节NADPH氧化酶组装中起关键作用。
