Kinetics, Regulation, And Mechanisms Of Biochemical Reac

生化反应的动力学、调控和机制

• 批准号: 6541587
• 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/6541587
• 关键词: actins; apoptosis; biological signal transduction; calcium flux; cell line; chemical kinetics; cysteine endopeptidases; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; epidermal growth factor; free radical oxygen; glutathione; glycation; hydrogen peroxide; membrane activity; nitric oxide; nitric oxide synthase; oxidative stress; phospholipase C; phosphorylation; protein tyrosine kinase; superoxide dismutase; thioredoxin

Free radical and reactive oxygen species (ROS) play an important role in the etiology and/or progression of a number of diseases and in aging as well as in signal transduction. To elucidate mechanisms by which free radicals and ROS are generated and exert such diverse biological effects, investigators in the Section on Metabolic Regulation carried out the following studies: Growth factors are known to induce transient generation of ROS and free radicals in non-phagocytic cells followed by elevation of tyrosine-phosphorylated and glutathionylated proteins. When A431 cells were treated with EGF, the ROS generated caused an elevation of the oxidized glutathione to total glutathione pool from the basal level of 5% to 12%. However, this shift towards the more oxidized state paradoxically leads to a net deglutathionylation of a 42 kDa protein, identified as actin. Mass spectrometric analysis revealed that the glutathionylated site is cysteine-374. In vitro studies showed that deglutathionylation of the G-actin led to a 6-fold increase in the rate of polymerization. This is in agreement with the observed 12% increase in F-actin content 5 min after EGF treatment and the F-actin was found in the periphery of the cells, suggesting that in response to growth factor actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by thioltransferase, since Cd(II), an inhibitor of thioltransferase, inhibits intracellular actin deglutathionylation at 2 ?M, comparable to its half-saturation point in vitro and mass spectral analysis showed efficient transfer of glutathione from immobilized glutathionylated actin to thioltransferase. Previously, we showed that in DT40 cells hydrogen peroxide induced both tyrosine phosphorylation of phospholipase C gamma 2 (PLC gamma 2) and activation of phosphatidylinositol 3-kinase (PI3K) and the PI3K-specific inhibitor, Wortmannin, partially inhibited the hydrogen-peroxide mediated Ca(II) release without affecting the tyrosine phosphorylation of PLC gamma 2. Overexpression of Bruton?s tyrosine kinase (Btk), which is activated by hydrogen peroxide, can overcome the effects of Wortmannin, a property unique to hydrogen peroxide-mediated effects. The role of Btk in regulating Ca(II) signaling was investigated using Btk-deficient DT40 cells and the transfectant expressing the wild-type Btk or Btk mutants at its kinase (R25E), Src homolog 2 (R307A), or pleckstrin homolog (R28C) domain. The overall results are consistent with the notion that functional SH and PH domains are required to form a complex with PLC gamma 2 through an adaptor protein, BLNK, in order to position Blk, PLC gamma 2, and its substrate, phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLC gamma 2 and to maximize its catalytic efficiency for IP3 production. Since tyrosine phosphorylation of signaling molecules is a balanced result between protein tyrosine kinases and protein tyrosine phosphatases, the role of tyrosine phosphatase CD45, which constitutes nearly 40% of the total protein tyrosine phosphatase activity in DT40 cells, in oxidative stress signaling was studied. Almost 90% of the phosphatase activity was rapidly inactivated upon hydrogen peroxide treatment. In comparison, hydrogen peroxide-induced Ca(II) mobilization was impaired in CD45-deficient DT40 cells. However, hydrogen peroxide induced tyrosine phosphorylation of PLC gamma 2 and PI3K activation appeared intact in CD45-deficient cells. This suggests that CD45, in addition to its phosphatase activity, has a positive role in oxidative stress signaling. During the glycation reaction between reducing sugars and free amino groups of proteins, alpha-dicarbonyl compounds, such as glyoxal, methylglyoxal, and deoxyglucosones, are produced. These compounds are more reactive than their parent sugars for reacting with amino groups of proteins to form inter-and intra-molecular cross-links of proteins and stable advanced end products that are known to accumulate with aging, diabetes mellitus, Alzheimer's disease, and other diseases. We investigated the structure and redox properties of cross-linked amino acids and proteins produced by glyoxal. Model reactions between glyoxal or glycolaldehyde and the amino acids, alanine or N-alpha-acetyl-lysine, produced free radicals. The structure of this radical was identified by EPR spectroscopy as N-substituted pyrazinium radical cation, which was formed by cross-linking two amino acids. Glycation of BSA by these carbonyl compounds also generated stable protein-bound free radical species, probably the N-substituted pyrazinium radical cation as observed with amino acids. The glycated protein reduced ferricytochrome c to ferrocytochrome c, which was accompanied by a large increase in the EPR signal amplitude of the protein-bound free radical cation. In addition, the glycated protein also catalyzed the oxidation of ascorbate. These results indicate that protein glycation generates active centers for catalyzing one electron redox reactions. One of the active centers generated by glyoxal is the cross-linked N-substituted pyrazine and its radical cation. Serum deprivation or treatment with 1-methyl-4-phenylpyridinium (MPP) caused human neuroblastoma cells (SH-SY5Y) to undergo apoptosis since either of these treatments produced an elevation in the hydroxyl radicals, malondialdehyde and 4-hydroxy-2-nonenal, which led to mitochondria-mediated apoptosis. We showed that thioredoxin (Trx) in the submicromolar range protected SH-SY5Y cells from apoptosis via a multiphasic mechanism that includes the suppression of cytochrome C release and the induction of Mn-superoxide dismutase and Bcl-2. When SH-SY5Y cells were subjected to 2 hours of nonlethal preconditioning stress, it caused a hormesis effect due to the induction of Trx. The effectiveness of Trx-mediated hormesis against oxidative stress-induced apoptosis is striking. It produced a 30-fold shift in LD50 in the MPP-induced neurotoxicity. The preconditioning induction of Trx can be attributed to the upregulation of neuronal nitric oxide synthase.

自由基和活性氧（ROS）在许多疾病的病因和/或进展、衰老以及信号转导中发挥重要作用。为了阐明自由基和活性氧的产生机制并发挥如此多样的生物学效应，代谢调控部分的研究者进行了以下研究：