OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

描述：(改编自申请者摘要)：许多环境 毒物、药物和滥用物质通过以下途径发挥作用 活性氧物种。许多疾病和中毒事件都与蛋白质有关 氧化、功能失调的蛋白分解、聚集、交联化和 积累。由于许多环境毒素、药物、除草剂、杀虫剂、 慢性退行性疾病导致氧化和/或 泛素化蛋白(可能是由于核心20S的功能障碍 蛋白酶体)，我们的结果应该具有广泛的健康意义。我们的远大， 长期目标是测试20S蛋白酶体复合体的理论 (没有19或我是调节器)选择性地识别和降级 哺乳动物细胞中的氧化修饰蛋白质。我们提出了氧化性的 修饰的蛋白质在体内不是泛素化的，但这种氧化会导致 暴露出直接与20S蛋白酶体结合的疏水斑块。这 选择性蛋白分解可防止受损蛋白质的积累，这将 否则会威胁到细胞功能和/或生存能力。我们的新力学研究 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20S蛋白酶体，20S蛋白酶体 绑定到其19s调节器(称为26S蛋白酶体的复合体)，以及20s 与11S调节因子结合的蛋白酶体(称为免疫蛋白酶体)。我们会 检测泛素依赖性并确定其氧化修饰(无 泛素“标记”)足以进行选择性蛋白分解。自.以来 泛素化系统和26S蛋白酶体是细胞周期所必需的 任何一种系统的进展和有丝分裂、缺失突变都是致命的。新的 我们现在要构建的Tet-off条件性调控细胞系将提供 无价的永久工具，将在未来许多年内使用。1.测试 假设核心20S蛋白酶体是降解蛋白质所必需的 氧化蛋白质。我们将构建永久性的WL-38人肺成纤维细胞 条件性调节(Tet-of)反义表达的细胞系 C5核心蛋白酶体必需亚基的序列，以便提供一种 用于测试核心20S蛋白酶体参与程度的全新工具 在体内降解氧化损伤的蛋白质。这些研究将 得到新改进的反义吗啡的辅助实验的支持 针对20S蛋白酶体CS亚基等亚基的寡核苷酸。我们 还将研究更多改进的细胞特定的直接蛋白酶体抑制剂 Lactacystin，Clastro lactacystin b-lacone，和NLVS。2.检验假设 泛素结合不是氧化降解的重要因素 蛋白质。Tet-Off第二代细胞系的条件调控表达 泛素激活EL酶的反义序列将用于 测试氧化的泛素结合的重要性(或无关性) 蛋白质。我们还将研究ts2O突变体中的蛋白质氧化和蛋白质分解。 在EL酶中含有温度敏感突变，使用非常短的 在不允许的温度或接触禁止的温度下灭活暴露， 融合培养，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，泛素醛将被 被测试为抑制剂。为了进一步测试氧化蛋白质的泛素化，我们 将使用谷胱甘肽-琼脂糖固定化-S5a(S5a是26S蛋白酶体 结合多泛素化蛋白的亚基)，并将S5a添加到细胞中 用于隔离任何泛素-蛋白质结合物并防止其 退化。来检验19s和11s蛋白酶体都不存在的假设 氧化蛋白质的降解需要调节剂。我们会 构建Tet-Off调控的第三株人成纤维细胞系 P56必需ATPase亚单位反义序列的表达 19S调节器，以测试I 9S调节器的重要性(或无关性) 氧化蛋白质降解的复合体。20S蛋白酶体复合体 不会在这些细胞中受到影响，但26S复合体的水平将受到影响 逐渐消沉。我们还将研究反义吗啡的作用。 针对p56亚单位和基本PA的寡核苷酸 蛋白酶体11S调节子的28α亚基。抗体抑制和 将在细胞提取液中研究I9S和1LS调节剂的免疫沉淀。 和纯化的26S和‘免疫蛋白酶体。

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.