KINETICS, REGULATION, AND MECHANISMS OF BIOCHEMICAL REACTIONS

生化反应的动力学、调控和机制

• 批准号: 6290350
• 负责人: P. BOON Chock
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290350
• 关键词: amyotrophic lateral sclerosis; biological signal transduction; calcium flux; chemical kinetics; electrochemistry; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; free radicals; hydrogen peroxide; isozymes; membrane activity; nitric oxide; oxidative stress; phospholipase C; phosphorylation; protein kinase; protein tyrosine kinase; superoxide dismutase; ubiquitin

Reactive oxygen species (ROS) have been implicated as signal transducers that cause an elevation of tyrosine phosphorylated proteins in non-phagocytic cells. This elevation can be achieved by inactivating protein tyrosine phosphatases (PTPs). We and others have shown in vitro that the highly abundant PTP, PTP-1B, is inactivated by ROS via oxidation of its catalytic site C215. We found that superoxide radical anion is a kinetically more efficient and chemically more specific oxidant than hydrogen peroxide for inactivating PTP-1B. The initial oxidized product, the C215 sulfenic derivative, can easily be oxidized further to its irreversible derivative, a reaction that can be prevented by glutathionylation. The S-glutathionylated PTP-1B can then be reactivated by thiotransferase. Thus, a signal transduction mechanism mediated by superoxide radical anion and the participation of glutathione is proposed for the regulation of PTP-1B. This mechanism is supported by the in vivo demonstration that glutathionylated PTP-1B at C215 is formed in A431 cells when they were treated with epidermal growth factor. In addition, the superoxide level is regulated by Mn(II)-superoxide dismutase (MnSOD). We found that a phorbol ester, TPA, induces human MnSOD gene via a protein kinase C-catalyzed phosphorylation of a CREB-1/ATF-1-like factor and not via known redox- sensitive transcription factors, such as NF-kappa-B or AP-1. Hydrogen peroxide has been shown to induce intracellular calcium release through the activation of phospholipase C-gamma-2 (PLC-gamma-2) via both tyrosine phosphorylation of PLC-gamma-2 and activation of phosphatidylinositol 3-kinase. - protein phosphatase, free radical, oxidative stress, fast reaction, vesicles, electroporation, EPR, glycation

活性氧（ROS）被认为是引起非吞噬细胞中酪氨酸磷酸化蛋白升高的信号转导子。这种升高可以通过使蛋白酪氨酸磷酸酶（PTP）失活来实现。我们和其他人已经在体外证明，高丰度的PTP，PTP-1B，通过氧化其催化位点C215被ROS灭活。我们发现，超氧阴离子自由基是一种动力学上更有效的和化学上更具体的氧化剂比过氧化氢灭活PTP-1B。最初的氧化产物，C215次磺酸衍生物，可以很容易地被进一步氧化成其不可逆的衍生物，这是一种可以通过谷胱甘肽化来防止的反应。然后，S-谷胱甘肽化PTP-1B可以被硫转移酶重新激活。因此，超氧阴离子自由基介导的信号转导机制和谷胱甘肽的参与提出的PTP-1B的调节。这一机制得到了体内实验的支持，即当A431细胞用表皮生长因子处理时，在C215处形成谷胱甘肽化PTP-1B。此外，超氧化物水平由Mn（II）-超氧化物歧化酶（MnSOD）调节。我们发现佛波醇酯TPA通过蛋白激酶C催化的CREB-1/ATF-1样因子的磷酸化而不是通过已知的氧化还原敏感性转录因子如NF-κ-B或AP-1诱导人MnSOD基因。已显示过氧化氢通过磷脂酶C-γ-2（PLC-γ-2）的酪氨酸磷酸化和磷脂酰肌醇3-激酶的活化来活化PLC-γ-2，从而诱导细胞内钙释放。- 蛋白磷酸酶，自由基，氧化应激，快速反应，囊泡，电穿孔，EPR，糖化