Hydrogen Peroxide As Intracellular Messenger

越来越多的证据表明，H2 O2作为细胞内信使介导各种细胞功能，包括增殖，分化，凋亡和衰老，当以低量和受控的方式产生时。了解H2 O2的细胞内信使功能，需要研究受体占据如何促进H2 O2的产生，以及H2 O2在完成其使命后如何被消除。 在用生长因子刺激的细胞中产生H2 O2需要激活磷脂酰肌醇3-激酶（PI 3 K）和小GT3激酶Rac。我们证明，betaPix，一个鸟嘌呤核苷酸交换因子Rac（Rac-GEF），和Nox 1，一个蛋白质相关的gp 91 phox（Nox 2）的吞噬细胞，有助于生长因子诱导的生产H2 O2在非吞噬细胞系。BetaPix被证明与Nox 1的COOH末端区域组成性相关，而Nox 1的该区域不结合Vav 1，另一个Rac-GEF。Rac 1也被证明结合到COOH-末端区域的Nox 1在生长因子依赖性的方式。生长因子诱导的Rac 1激活和H2 O2的产生都被RNA干扰完全阻断在细胞中耗尽betaPix。pleckstrin的同源性和亮氨酸拉链结构域的betaPix，介导bPix激活的产品PI 3 K的行动和betaPix同源二聚体，分别是必不可少的生长因子诱导的过氧化氢的生产。此外，Nox 1的COOH末端片段的表达完全抑制生长因子诱导的H2 O2产生。这些结果表明，在生长因子刺激的细胞中H2 O2的产生是由PI 3 K，betaPix和Rac 1的顺序激活介导的，后者然后结合到Nox 1，以刺激电子流从NADPH到氧分子。 过氧化物氧还蛋白I（Prx I）通过硫氧还蛋白（Trx）和Trx还原酶，利用来源于NADPH的还原当量催化H2 O2的还原。该二聚体酶的催化循环包括Cys 51-SH被H2 O2特异性氧化为Cys 51-SOH，在成对亚基的Cys 51-次磺酸和Cys 172-SH之间形成二硫化物，以及通过Trx还原分子间二硫化物。通过跟踪Prx依赖性NADPH氧化的荧光定量法，我们观察到Prx活性随时间逐渐降低。衰减的活动是一致的转换Prx我更酸性的物种，通过二维凝胶电泳评估。质谱分析和对Cys突变体的研究确定，pI的这种变化是由于催化位点Cys 51-SH选择性氧化为Cys-SOOH。因此，在催化过程中作为中间体产生的次磺酸Cys 51似乎偶尔进一步氧化为亚磺酸状态，这不能被Trx逆转。单独存在H2 O2不足以导致活性位点Cys氧化成磺酸。相反，完整的催化成分（H2 O2，Trx，Trx还原酶和NADPH）的存在是必要的，这表明只有当Prx I参与催化循环时才会发生这种过度氧化。同样，突变体Prx I，其中Cys 172被Ser取代，在完全反应混合物存在下不经历过氧化，因为突变体没有催化活性。超氧化的突变体酶不仅需要过氧化氢，但也催化支持硫醇，二硫苏糖醇。动力学分析的Prx我失活的存在下，一个稳定的状态，低水平（<1 μ M）的H2 O2表明，Prx我是超氧化在每营业额在30？C.在正常条件下培养的HeLa细胞中检测到过度氧化的Prx I，并且当向培养基中施加H2 O2时，Prx I以浓度依赖性方式增加。这种过度氧化可能导致H2 O2积累，从而导致细胞死亡。 以前，我们报道过从酵母中纯化的Prx在催化过程中很容易失活（1）。我们推测，如果反应中间体的次磺酸部分在与Cys 172形成二硫键之前被H2 O2进一步氧化为亚磺酸（Cys-SO 3 H），则会发生这种失活（1）。测定混合物中包含的Trx或DTT不能还原亚磺酸。最近，Mitsumoto等人（29）使用二维聚丙烯酰胺凝胶电泳（2-D凝胶）比较了细胞暴露于H2 O2前后人脐静脉内皮细胞中的蛋白质。在H2 O2处理的细胞中，一些蛋白质，包括Prx I和Prx II表现出改变迁移与降低等电点pH值（pI）一致，这表明这种氧化失活也可能发生在细胞中。然而，这些酸性Prx酶没有详细表征。我们现在已经研究了H2 O2对人Prx I失活的机制。在这里，我们证明，酶的失活和伴随的2-D凝胶上的Prx的酸位移实际上是由于活性位点的半胱氨酸的亚磺酸（Cys-SO2 H）的转换。此外，我们观察到，只有那些活跃参与催化循环的Prx分子容易受到氧化失活的影响。

There is increasing evidence that H2O2 serves as an intracellular messenger mediating various cell functions including proliferation, differentiation, apoptosis, and senescence, when produced in low amount and in a controlled fashion. Understanding the intracellular messenger function of H2O2 calls for studies of how receptor occupation elicits the production of H2O2 and how H2O2 is eliminated after the completion of its mission. The generation of H2O2 in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. We demonstrated that both betaPix, a guanine nucleotide exchange factor for Rac (Rac-GEF), and Nox1, a protein related to gp91phox (Nox2) of phagocytic cells, contribute to the growth factor-induced production of H2O2 in nonphagocytic cell lines. BetaPix was shown to be constitutively associated with the COOH-terminal region of Nox1, whereas this region of Nox1 did not bind Vav1, another Rac-GEF. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Both growth factor-induced Rac1 activation and H2O2 production were completely blocked in cells depleted of betaPix by RNA interference. The pleckstrin homology and leucine zipper domains of betaPix, which mediate bPix activation by products of PI3K action and betaPix homodimerization, respectively, were essential for growth factor-induced H2O2 production. Moreover, expression of a COOH-terminal fragment of Nox1 completely inhibited growth factor-induced H2O2 generation. These results suggest that H2O2 production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, betaPix, and Rac1, the latter of which then binds to Nox1 to stimulate electron flow from NADPH to oxygen molecules. Peroxiredoxin I (Prx I) catalyzes the reduction of H2O2 with the use of reducing equivalents derived from NADPH through thioredoxin (Trx) and Trx reductase. The catalytic cycle of this dimeric enzyme includes the specific oxidation of Cys51-SH by H2O2 to Cys51-SOH, the formation of a disulfide between the Cys51-sulfenic acid and Cys172-SH of the paired subunit, and the reduction of the intermolecular disulfide by Trx. By following Prx-dependent NADPH oxidation spectrophotometrically, we observed that Prx activity decreases gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by 2-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys51-SH to Cys-SOOH. Thus, the sulfenic Cys51 generated as an intermediate during catalysis appeared to undergo occasional further oxidation to the sulfinic state, which cannot be reversed by Trx. The presence of H2O2 alone was not sufficient to cause oxidation of the active site Cys to sulfnic acid. Rather, the presence of complete catalytic components (H2O2, Trx, Trx reductase, and NADPH) was necessary, indicating that such hyper-oxidation occurred only when Prx I was engaged in the catalytic cycle. Likewise, a mutant Prx I, in which Cys172 was replaced by Ser, did not undergo hyper-oxidation in the presence of a complete reaction mixture because the mutant was not catalytically active. Hyper-oxidation of the mutant enzyme required not only H2O2 but also a catalysis-supporting thiol, dithiothreitol. Kinetic analysis of Prx I inactivation in the presence of a steady-state, low level (<1 microM) of H2O2 indicated that Prx I was hyper-oxidized at a rate of 0.072% per turnover at 30?C. Hyper-oxidized Prx I was detected in HeLa cells cultured under normal conditions and increased in a concentration dependent-manner when H2O2 was applied to the culture media. Such hyper-oxidation is likely to result in H2O2 accumulation and thereby contribute to cell death. Previously, we reported that Prx purified from yeast is readily inactivated during catalysis (1). We speculated that such inactivation occurred if the sulfenic acid moiety of the reaction intermediate was further oxidized by H2O2 to sulfinic acid (Cys- SO3H) before disulfide formation with Cys172 could occur (1). Sulfinic acid cannot be reduced by the Trx or DTT included in the assay mixture. Recently Mitsumoto et al. (29) used two-dimensional polyacrylamide gel electrophoresis (2-D gel) to compare proteins in human umbilical vein endothelial cells before and after exposure of cells to H2O2. In H2O2-treated cells, a number of proteins including Prx I and Prx II demonstrated altered migration consistent with decreased isoelectric pH (pI), suggesting that such oxidative inactivation might also occur in cells. However, these acidic Prx enzymes were not characterized in detail. We have now investigated the mechanism of human Prx I inactivation by H2O2. Here, we demonstrated that the enzymatic inactivation and concomitant acidic shift of Prx on 2-D gels are due in fact to the conversion of the active site cysteine to sulfinic acid (Cys- SO2H). Furthermore, we observed that only those Prx molecules actively engaged in the catalytic cycle are vulnerable to oxidative inactivation.