Oxygen Radical Toxicity and Protein Degradation

描述（由申请人提供）：蛋白质是许多环境毒物氧化修饰的主要目标。它们失去了功能和结构，必须被蛋白水解降解，否则它们将聚集并形成交联的细胞包涵体。我们已经证明了蛋白酶体在氧化蛋白解毒中的主要作用。现在我们发现蛋白酶体在动态控制下，交换调节因子和催化亚基，并在适应轻度、急性应激时表现出从头合成。我们的长期目标是了解蛋白酶体促进基础和诱导氧化应激抵抗的机制，这种解毒系统对人体健康的贡献，以及衰老如何损害这种抵抗。我们的具体目的是验证以下假设：1)将人HBE1细胞或原代人NHBE细胞暴露于过氧化氢（H2O2）中，导致Ecm29催化的26S蛋白酶体瞬间可逆分解，HSP70稳定19S调节因子。26S蛋白酶体的解离阻止Nrf2降解，导致Nrf2积累、磷酸化和核易位；2)将HBE1人细胞或原代人NHBE细胞暴露于轻度适应剂量的H2O2中，可使Nrf2结合20S蛋白酶体亚基基因的ARE（或EpRE）序列和Pa28?？并在H2O2暴露后5-10小时内上调表达。Irf-1和/或NF？B转录因子同时结合到三个免疫蛋白酶体基因的上游区域，上调免疫蛋白酶体和Pa28？的表情;3)秀丽隐杆线虫和黑腹线虫对H2O2的适应需要20S蛋白酶体和Pa28？通过Nrf2的skn-1和cnc同源物表达；4)在我们的高氧模型中，衰老的HBE1和原代NHBE细胞适应氧化应激的能力下降，非分裂的HBE1和NHBE细胞以及来自老年人类供体的NHBE细胞加速衰老，部分原因是Nrf2和Irf-1/NF？蛋白酶体的B信号，Pa2?？和免疫蛋白酶体合成。h2o2诱导Pa28？而蛋白酶体通过Nrf2的同源物skin -1和cnc-C，也会在线虫和黑胃线虫的衰老过程中衰退，从而导致与年龄相关的整体抗逆性和适应性下降。

DESCRIPTION (provided by applicant): Proteins are major targets of oxidative modification by many environmental toxicants. They lose function & structure, and must be proteolyticaly degraded or they will aggregate and form cross- linked cellular inclusion bodies. 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, and exhibiting de novo synthesis in adaptation to mild, acute stress. Our Long-term Goals are to understand the mechanisms by which Proteasome contributes to basal and inducible oxidative stress resistance, the contributions of this detoxification system to human health, and how aging can compromise such resistance. Our Specific Aims are to test the following hypotheses: 1) Exposure of human HBE1 cells or primary human NHBE cells to hydrogen peroxide (H2O2), causes transient and reversible disassembly of 26S Proteasomes catalyzed by Ecm29, with stabilization of 19S regulators by HSP70. Dissociation of 26S Proteasomes prevents Nrf2 degradation, leading to Nrf2 accumulation, phosphorylation, and nuclear translocation; 2) Exposure of HBE1 human cells or primary human NHBE cells to a mild adaptive dose of H2O2, causes Nrf2 to bind to ARE (or EpRE) sequences of 20S Proteasome subunit genes and Pa28??? genes, and up-regulate their expression within 5-10 hours after H2O2 exposure. The Irf-1 and/or NF?B transcription factors simultaneously bind to upstream regions of the three Immunoproteasome genes, and up-regulate Immunoproteasome and Pa28?? expression; 3) Adaptation of C. elegans and D. melanogaster to H2O2 requires 20S Proteasome and Pa28? expression, via the skn-1 and cnc-C homologs of Nrf2; 4)The capacity to adapt to oxidative stress declines in senescent HBE1 and primary NHBE cells, in our model of hyperoxia-accelerated aging of non-dividing HBE1 and NHBE cells, and in NHBE cells from older human donors, partly due to diminished Nrf2 and Irf-1/NF?B signaling of Proteasome, Pa2???, and Immunoproteasome synthesis. H2O2-induced expression of Pa28? and Proteasome, through Nrf2 homologs skn-1 and cnc-C, also decays during aging of C. elegans and D. melanogaster, contributing to age-related decline in overall stress resistance and adaptability.