OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

描述：（改编自申请人的摘要）：许多环境 毒物、药物和滥用物质通过以下途径发挥作用： 活性氧许多疾病和中毒事件都涉及蛋白质 氧化、功能失调的蛋白水解、聚集、交联和 积累由于许多环境毒素，药物，除草剂，杀虫剂， 和慢性退行性疾病引起氧化和/或 泛素化蛋白（可能是由于核心20 S 蛋白酶体），我们的结果应该具有广泛的健康意义。我们的布罗德， 长期目标是检验20 S蛋白酶体复合物 （不含19 S或11 S调节剂）选择性识别和降解 哺乳动物细胞中的氧化修饰蛋白质。我们认为， 修饰的蛋白质在体内不是泛素化的，但氧化引起的是 暴露直接与20 S蛋白酶体结合的疏水斑块。这 选择性的蛋白水解防止了受损蛋白的积累， 否则会威胁细胞功能和/或生存力。我们新的机械研究 将测试解毒所需的蛋白酶体的确切形式， 氧化蛋白质我们将测试核心的20 S蛋白酶体， 与其19 S调节器（一种称为26 S蛋白酶体的复合物）结合，20 S 与11 S调节器结合的蛋白酶体（称为“免疫蛋白酶体”）。我们将 测试泛素依赖性并确定其氧化修饰（没有 泛素“标记”）足以进行选择性蛋白水解。以来 泛素化系统和26 S蛋白酶体是细胞周期所必需的 进展和有丝分裂，任何系统的缺失突变体都是致命的。新 我们现在构建的Tet-off条件调控细胞系将提供 宝贵的永久性工具将在未来许多年使用。1.测试 核心20 S蛋白酶体是降解蛋白质所必需的假设 氧化蛋白质。我们将构建一个永久的WI-38人肺成纤维细胞， 具有条件性调节（Tet-of）的反义核酸表达的细胞系 序列与C5核心蛋白酶体必需亚基连接，以提供一种 一种全新的工具，用于测试核心20 S蛋白酶体的参与 在体内氧化损伤蛋白质的降解中。这些研究将 得到了新改进的反义吗啉代辅助实验的支持 针对20 S蛋白酶体CS亚基和其它亚基的寡核苷酸。我们 还将研究更好的细胞渗透，直接蛋白酶体抑制剂 乳胞素、乳胞素b-内酯和NLVS。2.为了检验这一假设 泛素结合对氧化的降解不重要 Proteins.第二种Tet-off细胞系，其具有条件性调节的 将使用泛素激活E1酶的反义序列， 测试氧化的泛素缀合的重要性（或无关性） proteins.我们还将研究ts 2 O突变体中的蛋白氧化和蛋白水解 在El酶中具有温度敏感性突变，使用非常短的 使暴露于非允许温度下失活，或接触抑制， 融合培养物，以避免生长停滞效应。El的免疫沉淀 将被用作泛素化的对照，而泛素醛将被 作为抑制剂测试。为了进一步测试氧化蛋白的泛素化，我们 将使用谷胱甘肽-琼脂糖固定化-S5 a（S5 a是26 s蛋白酶体 亚基，其结合多泛素化蛋白），并将S5 a添加到细胞 提取物以螯合任何泛素-蛋白质缀合物并防止它们的 降解19 S和11 S蛋白酶体均不存在假说的验证 氧化蛋白质的降解需要调节剂。我们将 构建Tet-off调控的WI-38人成纤维细胞系 表达p56必需ATP酶亚基的反义序列， 19 S调节器，以测试I 9 S调节器的重要性（或无关性） 氧化蛋白质降解的复杂性。20 S蛋白酶体复合物 在这些细胞中不会受到影响，但26 S复合物的水平会受到影响。 逐渐沮丧。我们还将研究反义吗啉代的影响， 针对p56亚基和针对必需PA的寡核苷酸 蛋白酶体11 S调节子的28 α亚基。抗体抑制和 将在细胞提取物中研究19 S和11 S调节剂的免疫沉淀 并与纯化的26 S和免疫蛋白酶体结合。

DESCRIPTION: (Adapted from the Applicant's Abstract): Many environmental toxicants, medicinal drugs, and abuse substances exert their effects via reactive oxygen species. Many diseases and toxic events involve protein oxidation, dysfunctional proteolysis, aggregation, cross-linking, and accumulation. Since many environmental toxins, drugs, herbicides, pesticides, and chronic degenerative diseases cause an accumulation of oxidized and/or ubiquitinylated proteins (perhaps due to dysfunction of the core 20S proteasome), our results should have broad health significance. Our Broad, Long-Term Objective is to test the theory that the 20S proteasome complex (without 19S or I IS regulators) selectively recognizes and degrades oxidatively modified proteins in mammalian cells. We propose that oxidatively modified proteins are not ubiquitinylated in vivo, but that oxidation causes exposure of hydrophobic patches which directly bind to the 20S proteasome. This selective proteolysis prevents accumulation of damaged proteins which would otherwise threaten cell function and/or viability. Our new mechanistic studies will test the exact form of the proteasome required for detoxification of oxidized proteins. We will test the core 20S proteasome, the 20S proteasome bound to its 19S regulator (a complex called the 26S proteasome), and the 20S proteasome bound to the 11S regulator (called the 'immunoproteasome'). We will test for ubiquitin-dependence and determine it oxidative modification (without ubiquitin 'tagging') is sufficient for selective proteolysis. Since the ubiquitinylation system and the 26S proteasome, are required for cell-cycle progression and mitosis, deletion mutants of either system are lethal. The new Tet-off conditionally regulated cell lines we will now construct will provide invaluable permanent tools to be used for many years to come. 1. To Test the Hypothesis that the Core 20S Proteasome is Required for the Degradation of Oxidized Proteins. We will construct a permanent Wl-38 human lung fibroblast cell line with conditionally regulated (Tet-of) expression of an antisense sequence to the C5 core proteasome essential subunit, in order to provide an entirely new tool with which to test the involvement of the core 20S proteasome in the degradation of oxidatively damaged proteins in vivo. These studies will be supported by ancillary experiments with newly improved antisense morpholino oligonucleotides against the 20S proteasome CS subunit, and other subunits. We will also study much improved cell-permeant, direct proteasome inhibitors lactacystin, Clastro lactacystin b-lactone, and NLVS. 2. To Test the Hypothesis that Ubiquitin Conjugation is NOT Important for the Degradation of Oxidized Proteins. A second Tet-off cell line with conditionally regulated expression of an antisense sequence to the ubiquitin-activating El enzyme will be used to test the importance (or irrelevance) of ubiquitin conjugation of oxidized proteins. We will also study protein oxidation and proteolysis in ts2O mutants harboring a temperature sensitive mutation in the El enzyme, using very short inactivating exposures to the non-permissive temperature, or contact-inhibited, confluent cultures, to avoid growth-arrest effects. Immunoprecipitation of El will be used as a control for ubiquitinylation, and ubiquitin aldehydes will be tested as inhibitors. To further test ubiquitinylation of oxidized proteins, we will use glutathione-sepharose immobilized- S5a (S5a is a 26s proteasome subunit, which binds multiubiquitinylated proteins), and add S5a to cell extracts to sequester any ubiquitin-protein conjugates and prevent their degradation. To Test the Hypothesis that NEITHER the 19S NOR the 11S Proteasome Regulators are Required for the Degradation of Oxidized Proteins. We will construct a third WI-38 human fibroblast cell line with Tet-off regulated expression of an antisense sequence to the p56 essential ATPase subunit of the 19S regulator, to test the importance (or irrelevance) of the I 9S regulator complex in the degradation of oxidized proteins. The 20S proteasome complex will not be affected in these cells, but levels of the 26S complex will be gradually depressed. We will also study the effects of antisense morpholino oligonucleotides directed against the p56 subunit, and against the essential PA 28 alpha subunit of the proteasome 11S regulator. Antibody inhibition and immunoprecipitation of I 9S and 1lS regulators will be studied in cell extracts and with purified 26S and 'immunoproteasomes.