OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

描述(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，功能/活性松散，必须被蛋白质降解，否则它们将聚集在一起，形成交联的细胞包涵体。在这项资助的前几个周期中，我们已经展示了蛋白酶体在解毒氧化蛋白中的主要作用。现在我们发现，蛋白酶体处于动态控制下，在温和的急性应激下，交换调节器和催化亚基。了解蛋白酶体解毒的动态和诱导机制是我们的当务之急。我们广泛而长期的目标是确定氧化蛋白质选择性蛋白酶体蛋白分解的机制(S)，这个解毒系统对健康的贡献，以及在慢性或重复应激下蛋白酶体的损害。我们的具体目标是检验以下假设：1)在急性氧化应激中，20S蛋白酶体经历11 S(PA28)或PA200调节器，或PARP或HSP90的激活；而26S蛋白酶体经历HSP70促进的拆解和重组；2)蛋白酶体、免疫蛋白酶体和蛋白酶体调节因子的表达，是由急性氧化应激瞬时诱导的，允许氧化蛋白质更有效地降解；3)虽然急性(轻度)氧化应激瞬时提高蛋白酶体系统抵御氧化修饰蛋白的能力，但慢性或重复应激实际上损害了这种适应并增加了对环境毒素的长期易感性。过氧化氢将是研究的主要氧化剂。有限的比较研究将与过氧亚硝酸盐、次氯酸/次氯酸盐、羟基自由基、氧化还原循环苯二酚和高氧培养环境进行。蛋白酶体调节和亚单位交换的重要性将通过基因敲除突变、siRNA(或反义)、抗体沉淀和生化技术的组合进行测试。直接激活与亚基/调节子表达增加的对比将用转录和翻译抑制物进行测试。还将测试通过磷酸化或谷胱甘肽基化进行的直接调节。用于蛋白酶体亚基和调节子的结构性过表达细胞和条件转基因细胞将被用来模拟压力适应的机制，并检验我们的结论。我们的研究将通过改善对毒性机制的理解，特别是急性暴露与慢性或反复侮辱的对比，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.