OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

氧自由基毒性和蛋白质降解

• 批准号: 7910936
• 负责人: Kelvin J. A. Davies
• 金额: $ 29.46万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-04 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910936
• 关键词: 26S proteasome; Acute; Antibodies; Apoptosis; Biochemical; Calpain; Catalytic Domain; Cell Line; Cell Nucleus; Cells; Cellular Stress; Chronic; Comparative Study; Competence; Complex; Cytoplasm; Development; Drug Metabolic Detoxication; Environment; Exposure to; Genetic Transcription; Grant; Health; Heat-Shock Proteins 70; Heat-Shock Proteins 90; Hydrogen Peroxide; Hydrolase; Hydroxyl Radical; Hyperoxia; Hypochlorite; Hypochlorous Acid; Impairment; Inclusion Bodies; Knock-out; Life; Lysosomes; Metabolism; Modeling; Modification; Necrosis; Nitrogen; Nuclear; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Peroxonitrite; Phosphorylation; Poly(ADP-ribose) Polymerases; Precipitation; Predisposition; Process; Proteins; Proteolysis; Public Health; Quinones; Reactive Oxygen Species; Regulation; Research Personnel; Resistance; Role; Small Interfering RNA; Stress; System; Techniques; Testing; Tetanus Helper Peptide; Toxic Environmental Substances; Toxic effect; Toxin; Transgenic Organisms; Translations; Work; acute stress; biological adaptation to stress; crosslink; improved; inhibitor/antagonist; multicatalytic endopeptidase complex; mutant; overexpression; programs; protein degradation; response

DESCRIPTION (provided by applicant): Many environmental toxins are oxidants. Proteins, which are major targets of oxidative modification, loose function/activity and must be proteolyticaly degraded or they will aggregate, and form cross-linked cellular inclusion bodies. In previous cycles of this grant, we have shown a major role for the proteasome in detoxifying oxidized proteins. Now we find proteasome is under dynamic control, exchanging regulators, and catalytic subunits in mild, acute stress. Understanding dynamic and inducible mechanisms of detoxification by proteasome is our immediate priority. Our broad, long-term objective is to define the mechanism(s) by which oxidized proteins undergo selective proteasomal proteolysis, the contributions of this detoxification system to health, and the impairment of proteasome under chronic or repeated stress. Our specific aims are to test the following hypotheses: 1) During acute oxidative stress, 20S proteasome undergoes activation by 11 S(PA28) or PA200 regulators, or by PARP or HSP90; whereas 26S proteasome undergoes disassembly and reassembly facilitated by HSP70; 2) Expression of proteasome, immunoproteasome, and proteasome regulators, is transiently induced by acute oxidative stress, allowing more efficient degradation of oxidized proteins; and 3) While acute (mild) oxidative stress transiently improves the ability of the proteasomal system to protect against oxidatively modified proteins, chronic or repeated stress actually impairs such adaptation and increases long-term susceptibility to environmental toxins. Hydrogen peroxide will be the major oxidant studied. Limited comparative studies will be conducted with peroxynitrite, hypochlorous acid/hypochlorite, hydroxyl radical, redox cycling quinones, and a hyperoxic cultivation environment. The importance of proteasome regulators and subunit exchanges will be tested with a combination of knockout mutants, siRNA (or antisense), antibody precipitation, and biochemical techniques. Direct activation versus increased subunit/regulator expression will be tested with transcriptional and translational inhibitors. Direct regulation by phosphorylation or glutathionylation will also be tested. Constitutively overexpressing cells and tet-off conditional transgenic cells, for proteasome subunits and regulators, will be used to model mechanisms of stress-adaptation, and to test our conclusions. Our studies will contribute to public health through improved understanding of mechanisms of toxicity, especially acute exposure versus chronic or repeated insults.

描述（由申请人提供）：许多环境毒素都是氧化剂。蛋白质是氧化修饰的主要目标，功能/活性松散，必须进行蛋白水解降解，否则它们会聚集并形成交联的细胞包涵体。在本次资助的前几个周期中，我们已经展示了蛋白酶体在氧化蛋白质解毒中的主要作用。现在我们发现蛋白酶体处于动态控制之下，在轻度、急性应激下交换调节因子和催化亚基。了解蛋白酶体解毒的动态和诱导机制是我们的当务之急。我们广泛、长期的目标是确定氧化蛋白质经历选择性蛋白酶体蛋白水解的机制、该解毒系统对健康的贡献以及慢性或反复应激下蛋白酶体的损伤。我们的具体目标是测试以下假设： 1）在急性氧化应激期间，20S 蛋白酶体被 11 S（PA28）或 PA200 调节剂或 PARP 或 HSP90 激活；而 26S 蛋白酶体则在 HSP70 的促进下进行分解和重组； 2) 急性氧化应激短暂诱导蛋白酶体、免疫蛋白酶体和蛋白酶体调节剂的表达，从而更有效地降解氧化蛋白； 3) 虽然急性（轻度）氧化应激短暂地提高了蛋白酶体系统抵御氧化修饰蛋白质的能力，但慢性或重复的应激实际上会损害这种适应并增加对环境毒素的长期敏感性。过氧化氢将是研究的主要氧化剂。将针对过氧亚硝酸盐、次氯酸/次氯酸盐、羟基自由基、氧化还原循环醌和高氧培养环境进行有限的比较研究。蛋白酶体调节剂和亚基交换的重要性将通过敲除突变体、siRNA（或反义）、抗体沉淀和生化技术的组合进行测试。将使用转录和翻译抑制剂来测试直接激活与增加的亚基/调节子表达。还将测试磷酸化或谷胱甘肽化的直接调节。蛋白酶体亚基和调节因子的组成型过表达细胞和tet-off条件转基因细胞将用于模拟应激适应机制，并检验我们的结论。我们的研究将通过增进对毒性机制的了解，特别是急性暴露与慢性或反复损伤的了解，为公共卫生做出贡献。