Department of Pathology and Experimental Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2010 Apr 2;285(14):10281-90. doi: 10.1074/jbc.M109.084939. Epub 2010 Feb 5.
By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1-4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47(phox)/p67(phox), suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47(phox)/p67(phox), suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (K(d) = 58 +/- 12 nm). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes.
通过靶向信号蛋白中氧化还原敏感的氨基酸，NADPH 氧化酶（Nox）家族的酶将活性氧与生理过程联系起来。我们之前分析了 107 种 Nox 酶的序列，并鉴定了保守区域，这些区域预计在 Nox 结构或激活中具有重要功能。其中一个区域是细胞质 B 环，在 Nox1-4 中含有保守的多碱性区域。先前对 Nox2 的研究表明，B 环中的某些碱性残基对于 p47(phox)/p67(phox)的活性和易位很重要，这表明该区域参与亚基组装。然而，在不需要 p47(phox)/p67(phox)的 Nox4 中保守了该区域，这表明 B 环在 Nox 功能中具有额外的作用。在这里，我们通过突变 Nox4 B 环残基表明该区域对 Nox4 活性很重要。荧光偏振检测到 Nox4 B 环肽与脱氢酶结构域之间的结合（K(d) = 58 +/- 12 nm）。这种相互作用在 Nox4 R96E B 环中变弱，该突变也显著降低了全酶 Nox4 的活性。脱氢酶结构域的截断将 B 环结合位点定位到 NADPH 结合亚结构域的 N 端半部分。同样，Nox2 B 环与 Nox2 脱氢酶结构域结合，并且 Nox2 和 Nox4 的相互作用都依赖于 B 环的多碱性区域。这些数据表明 B 环对 Nox4 功能至关重要；我们提出 B 环通过与脱氢酶结构域结合，为 Nox 酶的跨膜和脱氢酶结构域之间提供了接口。
