OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

说明（由申请人提供）：许多环境毒素是氧化剂。蛋白质是氧化修饰的主要目标，其功能/活性松散，必须经过蛋白质水解降解，否则它们将聚集并形成交联的细胞包涵体。在之前的研究中，我们已经证明了蛋白酶体在氧化蛋白解毒中的重要作用。现在我们发现蛋白酶体处于动态控制之下，在轻度和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。我们广泛的长期目标是确定氧化蛋白进行选择性蛋白酶体蛋白水解的机制，这种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。我们的具体目的是验证以下假设：1)在急性氧化应激期间，20S蛋白酶体被11s （PA28）或PA200调节因子或PARP或HSP90激活；而26S蛋白酶体则在HSP70的促进下进行拆解和重组；2)蛋白酶体、免疫蛋白酶体和蛋白酶体调节因子的表达可在急性氧化应激下短暂诱导，从而更有效地降解氧化蛋白；3)急性（轻度）氧化应激可以短暂地提高蛋白酶体系统对氧化修饰蛋白的保护能力，而慢性或反复的应激实际上会损害这种适应能力，并增加对环境毒素的长期易感性。过氧化氢将是主要的氧化剂。有限的比较研究将在过氧亚硝酸盐、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下进行。蛋白酶体调节因子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。将用转录和翻译抑制剂测试直接激活与增加亚基/调节因子表达的关系。磷酸化或谷胱甘肽化的直接调节也将被测试。蛋白酶体亚基和调节因子的组成性过表达细胞和条件性转基因细胞将用于模拟应激适应机制，并验证我们的结论。我们的研究将通过提高对毒性机制的理解，特别是急性暴露与慢性或反复损伤的对比，为公众健康做出贡献。

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.