2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/654196摘要：細胞與組織勉力維持胞內之還原環境以協助粒腺體及內質網功能、去除活性自由基、及從事生物分子合成，真核生物於是演化出一套複雜的抗氧化系統含抗氧化物及抗氧化基因‧抗氧化基因的表現有賴於Nrf2轉錄因子及含Antioxidant response element (ARE) DNA序列於起動子的基因，正常狀態Nrf2蛋白與Keap1蛋白結合，導致滯留於細胞質且被降解‧適度的氧化壓力造成Keap1上半胱氨酸氧化及Keap1不活化，釋出Nrf2並伴隨著Nrf2磷酸化，導致Nrf2進核並與Maf蛋白結成而形成有功能之轉錄因子，其轉錄之抗氧化基因包括NAD(P)H:quinone oxidoreductase, glutathione S-transferase, heme oxgenase-1, glutathione peroxidase 3, glutamate cysteine ligase, and peroxiredoxin 1等‧但是仍存在著其它未知之偵測氧化壓力之機制，值得探索‧ 氧化壓力常伴隨著一些病理狀態如創傷、病毒感染、肥胖、糖尿病、心臟病、氣喘及神經退化疾病，而且也和正常生理有關如老化、特定細胞的分化及壓力反應中的訊息傳導‧活性自由基的作用對象以蛋白質之半胱氨酸殘基殘基為最重要，主要之氧化反應為亞硝基化(nitrosylation)、次磺酸化(sulfenylation) 及雙硫鍵形成‧我們已建立了純化及偵測上述半胱氨酸氧化修飾的方法並運用於一般生化分析及質譜分析‧本計畫擬探討一、細胞內有那些蛋白質會受到氧化修飾？二、氧化修飾如何影響這些蛋白質之功能、細胞分佈及交互作用對象，三、細胞在氧化壓力過後如何回復細胞內還原環境，四、運用上述知識於不同生物題目如病毒感染、細胞分化、細胞膜修補等，五、探討上述問題的過程中發展相關新穎技術‧如此我們可更瞭解蛋白質氧化修飾之作用、細胞氧化還原之調控、氧化壓力於細胞分化及細胞膜修補之角色，這些基礎知識有助於瞭解特定疾病的起因及發展‧ <br> Abstract: Cells and tissues maintain a reducing milieu inside the cells to sustain normal function in the mitochondria and endoplasmic reticulum, to scavenge reactive oxygen/nitrogen species (ROS/RNS), and to engage reductive biosynthesis. To avoid excessive ROS/RNS, a complex anti-oxidative defense system has evolved utilizing antioxidants and anti-oxidative enzymes. Regulated expression of anti-oxidative enzymes during oxidative stress is mostly mediated by antioxidant response elements (AREs) in the promoter of oxidant-sensitive genes and ARE-binding transcription factors, nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Under resting conditions, Nrf2 is localized in the cytoplasm where it is bound to the actin-binding protein Keap1 (Kelch-like ECH-associating protein 1) leading to its degradation. Following oxidative challenge, Nrf2 is released from Keap1 owing to multiple cysteine oxidation in Keap1 and/or by phosphorylation of Nrf2 by protein kinase C. These post-translational modifications allow free Nrf2 to translocate into the nucleus and associate with small Maf proteins. Genes containing AREs in their promoter regions including NAD(P)H:quinone oxidoreductase, glutathione S-transferase, heme oxgenase-1, glutathione peroxidase 3, glutamate cysteine ligase, and peroxiredoxin 1 are then up-regulated. However, other sensors which detect oxidation stress leading to activation of Nrf2 and manifestation of cellular responses remain to be discovered. Oxidative stress has been associated with the manifestation of pathophysiological conditions such as wounding, virus infection, aging, obesity, diabetes, cardiac disease, neurodegenerative diseases and asthma. In addition, ROS/RNS can mediate a number of physiological effects in cells such as adaptive changes in signaling in response to various stresses or during normal cell differentiation. Among the victims of untamed ROS/RNS, protein cysteine thiol is most prominent in its reactivity and also in its consequence following oxidation. Major types of reversible protein thiol modification by NO and related electrophiles include S-nitrosylation, sulfenic acid oxidation, and disulfide bond formation. We have set up and established methods for detection of endogenous S-nitrosylated, sulfenylated and thiolated proteins with compatibility with general biochemical methods such as SDS-PAGE, immunodetection, and mass spectrometry. More specifically, we wish to investigate 1) the types of proteins undergoing S-nitrosylation, sulfenic acid and disulfide oxidation through proteomics approach, 2) how oxidative modifications affect the function and localization of the target proteins, 3) how cells respond to exogenous oxidative stress in terms of immediate and long-term redox regulation mechanisms operating to counteract the manipulated oxidative stress, 4) the roles of redox regulation in cell differentiation and membrane wounding repair. And, in the processes of accomplishing the above aims we will develop novel techniques. The results lead to better understanding in the regulatory function of protein oxidation modification, mechanism of cell redox regulation, roles of oxidative stress in cell differentiation and membrane repair. The acquired knowledge is expected to be applied in the research of diseases associated with oxidative stress such as virus infection, cell wounding, cell apoptosis, diabetes and neurodegenerative diseases.Nrf2轉錄因子抗氧化系統雙硫鍵細胞分化細胞膜修補Nrf2thiolationsulfenic acidcell differentiationmembrane repair