OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

许多环境毒物、药物和滥用物质 它们通过氧自由基和相关的活性氧的毒性作用 物种。尽管对脂质过氧化进行了广泛的研究，但仍然很少 已知蛋白质氧化和可能的细胞保护作用 特殊的蛋白水解酶。反义技术的最新进展 寡脱氧核苷酸现在可以研究蛋白酶体核心蛋白水解物 完整细胞中的复合体。 我们广泛的、长期的目标是测试氧化的理论 细胞蛋白质被670 kDa识别和选择性降解 蛋白酶体核心复合体。有人提出，这种选择性的蛋白质分解 防止受损蛋白质的积累，否则就会 威胁细胞功能和/或生存能力。此外，还建议 某些细胞蛋白质特别容易被氧化。 因此，改装在他们的更替中起着主要作用。 我们的第一个特定目标将测试蛋白酶体防止 或最大限度地减少受损蛋白质的积累，保护细胞功能 和/或氢暴露对肝克隆9上皮细胞活性的影响 培养中的过氧化应激。在我们的克隆9肝细胞培养模型中 蛋白酶体水平可以通过长期暴露在反义分子中来调节 针对猪细小病毒起始密码子区域的寡核苷酸 蛋白酶体C2亚单位基因。在这种操纵之后，我们将揭露 细胞对过氧化氢的氧化应激反应。超氧化物，4- 在时间允许的情况下，也可以检测羟基壬烯醛和丙二醛。 针对抗病毒寡核苷酸的有限(筛查型)研究 其他蛋白酶体亚单位基因，以及一些新的 合成的蛋白酶体抑制剂，也将进行。详细 蛋白质氧化修饰的研究将包括二羟甲基酪氨酸 形成，羰基形成，蛋白质聚集，蛋白质交联， 蛋白质沉淀，蛋白质碎裂。单元格参数 需要测量的功能包括：生长率、克隆形成能力、蛋白质 合成和DNA合成。我们还将测试膜的完整性 和坏死性细胞死亡的相关方面，与不同的特征 细胞凋亡，或永久性生长停滞。 我们的第二个特定目标将检验Ezrin是AT之一的假设 暴露于Clone 9肝细胞中的至少6种选择性降解蛋白 并将寻求确定其他五人的身份 广泛降解的蛋白质。初步鉴定Ezrin为基础 与24个氨基酸的N-末端部分95%的序列同源性 我们降解的蛋白质之一(从2-D凝胶中提取)，现在必须是 用免疫印迹和放射免疫分析中的抗体进行了确认。这个 其他五种未知蛋白质将从2-D凝胶中回收并进行 到N-末端测序。如果发现新的序列，我们将 集中精力克隆相关基因，从筛选一只老鼠开始 肝脏c DNA文库。因为降解的蛋白质必须在下列情况下替换 氧化应激，我们将进行西部，北方和核奔跑 过氧化氢回收过程中转录和翻译的研究 曝光。

Many environmental toxicants, medicinal drugs, and abuse substances exert their toxic effects via oxygen free radicals and related active oxygen species. Despite extensive work on lipid peroxidation little is still known of protein oxidation and the possible cytoprotective role of specialized proteolytic enzymes. Recent advances with antisense oligodeoxynucleotides now permit study of the proteasome core proteolytic complex in intact cells. Our BROAD, LONG-TERM OBJECTIVE is to test the theory that oxidized cellular proteins are recognized and selectively degraded by the 670 kDa proteasome core complex. It is proposed that this selective proteolysis prevents the accumulation of damaged proteins which would otherwise threaten cell function and/or viability. It is further proposed that certain cellular proteins are particularly susceptible to oxidative modification which, therefore, plays a major role in their turnover. Our FIRST SPECIFIC AIM will test the hypothesis that proteasome prevents or minimizes accumulation of damaged proteins and preserves cell function and/or viability in liver Clone 9 epithelial cells exposed to hydrogen peroxide stress in culture. In our Clone 9 liver cell culture model proteasome levels can be modulated by prolonged exposure to an antisense oligodeoxynucleotide directed against the initiation codon region of the proteasome C2 subunit gene. Following such manipulation we will expose cells to the oxidative stress of hydrogen peroxide. Superoxide, 4- hydroxynonenal, and malonyldialdehyde may also be tested as time allows. Limited (screening-type) studies of oligodeoxynucleotides directed against other proteasome subunit genes, as well as studies of some newly synthesized proteasome inhibitors, will also be performed. Detailed studies of protein oxidative modification will include dityrosine formation, carbonyl formation, protein aggregation, protein cross-linking, protein precipitation, and protein fragmentation. Parameters of cell function to be measured are; growth rates, clonogenic viability, protein synthesis, and DNA synthesis. We will also test for membrane integrity and related aspects of necrotic cell death, versus various features of apoptosis, or permanent growth arrest. Our SECOND SPECIFIC AIM will test the hypothesis that ezrin is one of at least six selectively degraded proteins in Clone 9 liver cells exposed to H2O2 and will seek to determine the identities of the five other extensively degraded proteins. Tentative identification of ezrin is based on 95% sequence identity with a 24 amino acid long N-terminal portion of one of our degraded proteins (excised from 2-D gels) and must now be confirmed, using antibodies in Western blots and radioimmunoassays. The five other unknown proteins will be recovered from 2-D gels and subjected to N-terminal sequencing. Should novel sequences be discovered, we will concentrate on cloning the genes responsible, starting by screening a rat liver cDNA library. Since degraded proteins must be replaced following oxidative stress we will perform Western, Northern, and nuclear run-on studies of transcription and translation during recovery from H2O2 exposure.