Hydrogen Peroxide as Intracellular Messenger

据去年报道，过氧化氢还蛋白的活性部位半胱氨酸在催化过程中被选择性地氧化成半胱氨酸亚磺酸(Cys！VSO2H)，导致过氧化物酶活性失活。用35S对哺乳动物细胞的代谢标记表明，细胞暴露在过氧化氢中产生的过氧化氢还蛋白I的亚磺酸形式迅速还原为具有催化活性的硫醇形式。哺乳动物细胞的这种还原蛋白质亚磺酸的能力与普遍认为的亚磺酸状态的氧化是细胞中不可逆的过程相反。这种还原反应可能是一种修复氧化损伤蛋白质的机制，也可能代表了一种新型的循环修饰，通过这种修饰来调节各种蛋白质的功能。 我们已经鉴定并鉴定了一个14 kDa的人类硫氧还蛋白(TRX)相关蛋白，命名为TRP14。这种胞浆蛋白在所研究的所有组织和细胞类型中都有表达，通常比Trx1的表达数量更少。TRP14的生化特性表明，与Trx1类似，TRP14是一种二硫键还原酶；其活性部位半胱氨酸具有足够的亲核性，其氧化还原电位与其他细胞内的硫醇还原剂相似。然而，尽管TRP14减少了含有二硫键的小肽，但它并没有减少几种Trx1底物的二硫键，这表明TRP14和Trx1与不同的靶点相互作用。在HeLa细胞中，通过RNA干扰研究了TRP14与Trx1在肿瘤坏死因子-α信号转导中的可能作用。与Trx1缺失相比，部分缺失TRP14在更大程度上增强了肿瘤坏死因子-α诱导的IkBalpha的磷酸化和降解，以及随后的核因子-kB的激活。TRP14或Trx1的缺失在一定程度上增强了肿瘤坏死因子-α诱导的半胱氨酸天冬氨酸酶的激活和随后的细胞凋亡。然而，肿瘤坏死因子-α诱导的JNK和p38丝裂原活化蛋白激酶的激活可以通过去除TRP14而不是Trx1来促进。与Trx1不同，TRP14既不与ASK1的激酶活性相关，也不抑制ASK1的活性。这些结果表明，TRP14可能通过与不同于Trx1靶标的蛋白质相互作用来调节肿瘤坏死因子-α信号通路。 肿瘤坏死因子-α(TNF-α)可诱导三种丝裂原活化蛋白激酶(MAPK)的激活：c-Jun氨基末端激酶(JNK)、p38和细胞外信号调节激酶(ERK)。这种细胞因子还能诱导几种类型的活性氧的产生，包括过氧化氢。通过使用表达野生型或显性胞浆过氧化物酶过氧化物酶II阴性形式的HeLa细胞和缺乏该蛋白的小鼠胚胎成纤维细胞，我们评估了过氧化氢在肿瘤坏死因子-α激活MAPKs中的作用。体外激酶分析和针对活化的MAPK的抗体的免疫印迹分析表明，肿瘤坏死因子-α反应产生的H_2O_2增强了该细胞因子诱导的JNK和p38的激活，但抑制了ERK的激活。我们的结果还表明，胞浆中的过氧化还蛋白是肿瘤坏死因子信号通路的重要调节因子。

As reported last year, the active site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid (Cys!VSO2H) during catalysis, resulting in inactivation of peroxidase activity. Metabolic labeling of mammalian cells with 35S has now revealed that the sulfinic form of peroxiredoxin I produced during exposure of cells to H2O2 is rapidly reduced to the catalytically active thiol form. This ability of mammalian cells to reduce protein sulfinic acid is contrary to the general belief that oxidation to the sulfinic state is an irreversible process in cells. This reduction reaction might serve as a mechanism to repair oxidatively damaged proteins or represent a new type of cyclic modification by which the function of various proteins is regulated. We have identified and characterized a14-kDa human thioredoxin (Trx)-related protein designated TRP14. This cytosolic protein was expressed in all tissues and cell types examined, generally in smaller amounts than Trx1. Biochemical characterization of TRP14 suggested that, like Trx1, TRP14 is a disulfide reductase; its active site cysteine is sufficiently nucleophilic and its redox potential is similar to those of other cellular thiol reductants. However, although TRP14 reduced small disulfide-containing peptides, it did not reduce the disulfides of several Trx1 substrates, suggesting that TRP14 and Trx1 interact with distinct targets. The possible role of TRP14 in tumor necrosis factor-alpha (TNF-alpha) signaling was also studied in comparison with Trx1 by RNA interference in HeLa cells. Partial depletion of TRP14 augmented the TNF-alpha- induced phosphorylation and degradation of IkBalpha as well as the consequent activation of NF-kB to a greater extent than did Trx1 depletion. Deficiency of TRP14 or Trx1 enhanced TNF-alpha induced activation of caspases and subsequent apoptosis by similar extents. The TNF-alpha-induced activation of JNK and p38 mitogen-activated protein kinases, however, was promoted by depletion of TRP14 but not by that of Trx1. Unlike Trx1, TRP14 neither associated with nor inhibited the kinase activity of ASK1. These results suggest that TRP14 modulates TNF-alpha signaling pathways, probably by interacting with proteins distinct from the targets of Trx1. Tumor necrosis factor-alpha (TNF-alpha) induces the activation of all three types of mitogen-activated protein kinase (MAPK): c-Jun NH2-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). This cytokine also induces the production of several types of reactive oxygen species including H2O2. With the use both of HeLa cells expressing wild-type or dominant negative forms of the cytosolic peroxidase peroxiredoxin II and of mouse embryonic fibroblasts deficient in this protein, we evaluated the roles of H2O2 in the activation of MAPKs by TNF-alpha. In vitro kinase assays as well as immunoblot analysis with antibodies specific for activated MAPKs indicated that H2O2 produced in response to TNF-alpha potentiates the activation of JNK and p38 induced by this cytokine but inhibits that of ERK. Our results also suggest that cytosolic peroxiredoxins are important regulators of TNF signaling pathways.