Hydrogen Peroxide As Intracellular Messenger

过氧化氢是有丝分裂反应的细胞内介质。因此，细胞暴露于无毒浓度的H2O2刺激增殖，而接触抑制引起的生长停滞与细胞内H2O2浓度的降低有关。此外，细胞在响应生长刺激（如血小板衍生生长因子（PDGF））时短暂地产生H2O2，抑制这种作用会阻断这种刺激的下游信号传导。虽然H2O2与细胞内信号传递有关，但直接被H2O2靶向并传播信号的分子仍有待鉴定。蛋白质靶点不太可能特异性地结合H2O2。然而，由于与附近带正电的氨基酸相互作用，某些蛋白质半胱氨酸残基在中性pH下以硫代阴离子的形式存在，这些残基比质子化半胱氨酸更容易被H2O2氧化。含有电离半胱氨酸的蛋白质包括蛋白质酪氨酸磷酸酶家族的成员。PTEN是这个家族的一员，通过催化去除附着在PI肌醇环3'-羟基上的磷酸来逆转PI 3-激酶的作用。通过负向调节PI 3-激酶？在Akt信号通路中，PTEN是一个重要的肿瘤抑制因子。人类PTEN的必需半胱氨酸-124残基在活性位点口袋中被三个碱性氨基酸残基包围，细胞暴露于H2O2或PDGF也会产生3'-磷酸化磷酸肌苷（PIs），这是细胞存活和增殖的重要调节因子。细胞中PI的3′-磷酸化程度反映了PI 3-激酶和3′-磷酸酶PTEN作用的平衡。我们的研究结果表明，H2O2通过介导必需半胱氨酸-124的氧化和半胱氨酸-71的二硫残基形成，诱导PTEN的可逆失活。氧化酶被硫氧还蛋白重新激活。PTEN也被细胞中响应PDGF产生的H2O2氧化。我们的数据表明，响应PDGF和其他生长刺激而产生的H2O2对PTEN的可逆失活对于3'-磷酸化pi的积累是重要的，并且与某些病理条件相关的H2O2的不受控制的产生可能通过抑制PTEN功能来促进细胞增殖。如环核苷酸所示，在完成其功能后及时消除细胞内介质对细胞信号传导至关重要。因此，在受体占据后，第二信使的消除和产生都受到高度控制。对于H2O2来说尤其如此，它很容易转化为有害的羟基自由基。能够消除H2O2的酶包括过氧化氢酶和谷胱甘肽过氧化物酶以及过氧化物还蛋白（Prx）。没有证据表明过氧化氢酶和谷胱甘肽过氧化物酶这两种众所周知的酶的活性受到调控。此外，过氧化氢酶仅局限于过氧化物异构体中。Prx是一种新的过氧化物酶家族，存在于所有生物体内。所有Prx酶在氨基末端区域都含有一个保守的半胱氨酸残基，这是H2O2氧化的主要位点。哺乳动物Prx至少存在6种亚型，可分为3个亚群，即2-Cys亚群、非典型2-Cys亚群和1-Cys亚群。2-Cys成员，包括两个胞质酶Prx I和Prx II。Prx酶在各种细胞中过表达时，可有效降低血小板源性生长因子和肿瘤坏死因子α (tnf - α)刺激的细胞内产生的H2O2水平，抑制tnf - α诱导的NF-kappaB活化，阻断神经酰胺诱导的细胞凋亡，表明Prx酶通过去除H2O2作为信号级联反应的组成部分。然而，没有证据表明Prx酶的活性像许多其他细胞内信使代谢酶一样受到细胞环境变化的调节。我们注意到，来自人类、大鼠和小鼠的2-Cys Prx亚群成员的氨基酸序列都包含细胞周期蛋白依赖性激酶（Cdks）磷酸化的一致序列，(Ser/Thr)- pro - x -(Lys/Arg)，而Prx V和Prx VI不包含一致序列。假定的磷酸化位点Thr，对应于人类Prx I的Thr 90，位于所有4个2-Cys Prx成员的氨基末端保守的Cys下38个氨基酸。为了检测Cdk是否能磷酸化2-Cys Prx酶，我们将纯化的Prx I、II、III和IV用重组Cdc2激酶/cyclinB复合物进行激酶反应。prx1是最好的底物。为了便于监测Prx I在细胞中的磷酸化情况，我们用与Thr90磷酸化Prx I残基83和95之间的序列对应的13个残基磷酸化肽免疫兔子，制备了Prx I在Thr90位点磷酸化的特异性抗体。使用这种磷酸化特异性抗体，我们证明纯化的Prx I在Thr90位点被周期蛋白依赖激酶（Cdks）磷酸化，包括细胞分裂周期2 （Cdc2）激酶。在HeLa、HepG2、NIH3T3细胞中，Prx I的磷酸化发生在有丝分裂期，而不在间期。这些结果表明，Cdc2激酶（在有丝分裂期被激活的Cdk）在核膜被破坏后遇到Prx I并使其磷酸化。Cdc2激酶依赖的Prx I失活和由此产生的H2O2积累可能是细胞周期进展的重要过程。

Hydrogen peroxide is an intracellular mediator of the mitogenic response. Thus, exposure of cells to nontoxic concentrations of H2O2 stimulates proliferation, whereas growth arrest induced by contact inhibition is associated with a decrease in the intracellular concentration of H2O2. Furthermore, cells transiently produce H2O2 in response to growth stimuli such as platelet-derived growth factor (PDGF), and inhibition of this effect blocks downstream signaling by such stimuli. Although H2O2 is implicated in intracellular signaling, molecules that are directly targeted by H2O2 and thereby propagate the signal remain to be identified. It is unlikely that protein targets bind H2O2 specifically. However, certain protein cysteine residues exist as thiolate anions at neutral pH as a result of interaction with nearby positively charged amino acids, and these residues are more susceptible to oxidation by H2O2 than are protonated cysteines. Proteins that contain an ionized cysteine include members of the protein tyrosine phosphatase family. PTEN is a member of this family and reverses the action of PI 3-kinase by catalyzing the removal of the phosphate attached to the 3'-hydroxyl group of the PI inositol ring. By negatively modulating the PI 3-kinase?Akt signaling pathway, PTEN functions as an important tumor suppressor. The essential cysteine-124 residue of human PTEN is surrounded by three basic amino acid residues in the active site pocket Exposure of cells to H2O2 or to PDGF also results in the generation of 3'-phosphorylated phosphoinositides (PIs), which are important regulators of cell survival and proliferation. The extent of 3'-phosphorylation of PIs in a cell reflects the balance between the action of PI 3-kinase and that of the 3'-phosphatase PTEN. Our results show that H2O2 induces reversible inactivation of PTEN by mediating oxidation of the essential cysteine-124 and the formation by this residue of a disulfide with cysteine-71. The oxidized enzyme was reactivated by thioredoxin. PTEN was also oxidized by H2O2 produced in cells in response to PDGF. Our data suggest that the reversible inactivation of PTEN by H2O2 produced in response to PDGF and other growth stimuli is important for the accumulation of 3'-phosphorylated PIs, and that the uncontrolled generation of H2O2 associated with certain pathological conditions might contribute to cell proliferation by inhibiting PTEN function. As exemplified by cyclic nucleotides, timely elimination of intracellular mediator after completion of their functions is critical for cellular signaling. Thus, elimination as well as production of second messengers are process highly controlled following the occupancy of receptors This would seem especially true for H2O2, which is readily converted to deleterious hydroxyl radicals. Enzymes that are capable of eliminating H2O2 includes catalase and glutathione peroxidase, and peroxiredoxin (Prx). There are no evidence that the activities of the two commonly known enzymes catalase and glutathione peroxidase are regulated In addition, catalase is exclusively localized in the peroxisosmes. Prx is a novel family of peroxidases that are present in organisms from all kindoms. All Prx enzymes contain a conserved cysteine residue at the amino-terminal region, which is the primary site of oxidation by H2O2. Mammalian Prx exists as at least six isoforms, which can be divided into three subgroups, namely 2-Cys, atypical 2-Cys and 1-Cys subgroups . The 2-Cys members, which include two cytosolic enzymes Prx I and Prx II. When overexpressed in various cells, Prx enzymes efficiently reduced the intracellular level of H2O2 produced in the cells stimulated with platelet-derived growth factor and tumor necrosis factor-alpha (TNF-alpha), inhibited NF-kappaB activation induced by TNF-alpha, and blocked the apoptosis induced by ceramide, indicating that Prx enzymes serve as component of signaling cascades by removing H2O2. However, there is no evidence that the activity of Prx enzymes is regulated in response to changes in cellular environments as in the case of many other intracellular messenger metabolizing enzymes. We noticed that the amino acid sequences of the 2-Cys Prx subgroup members from human, rat, and mouse all contain the consensus sequence for phosphorylation by cyclin-dependnet kinases (Cdks), (Ser/Thr)-Pro-X-(Lys/Arg), whereas Prx V and Prx VI do not contain the consensus sequence. The putative phosphorylation site Thr, which corresponds to Thr 90 of human Prx I, is located 38 amino acids down from the amino-terminal conserved Cys for all four 2-Cys Prx members. To test whether Cdk can phosphorylate 2-Cys Prx enzymes, purified Prx I, II, III, and IV were subjected to kinase reaction by recombinant Cdc2 kinase/cyclinB complex in. Prx I was the far best substrate. To facilitate the monitoring of Prx I phosphorylation in cells, antibody specific to Prx I phosphorylated at Thr 90 was prepared by immunizing rabbits with a 13-residue phosphopeptide that corresponds to the sequence between residues 83 and 95 of Thr90-phosphorylated Prx I Using this phosphorylation-specific antibodies, we demonstrated that purified Prx I is phosphorylated at Thr90 by cyclin-dependent kinases (Cdks) including the cell division cycle 2 (Cdc2) kinase. The phosphorylation of Thr90 resulted in substantial reduction in the peroxiase activity of Prx I. In HeLa, HepG2, NIH3T3 cells Prx I phosphorylation occurrs in cells in mitotic phase but not in interphase. These results suggest that Cdc2 kinase, the Cdk that is activated in mitotic phase, encounters and phosphorylates Prx I after the nuclear envelop breaks down The Cdc 2 kinase-dependent inactivation of Prx I and the resulting accumulation of H2O2 are likely important processes for the progression of cell cycle.