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靶点。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重要作用。目前研究发现，蛋白酶体在适应轻度、急性应激的过程中，处于动态调控下，调节子和催化亚基相互交换，并表现出从头合成的特点。我们的长期目标是了解蛋白酶体有助于基础和诱导性氧化应激抵抗的机制，这种解毒系统对人类健康的贡献，以及衰老如何损害这种抵抗力。我们的具体目的是测试以下假设：1）将人HBE 1细胞或原代人NHBE细胞暴露于过氧化氢（H2 O2），引起由Ecm 29催化的26 S蛋白酶体的瞬时和可逆的分解，并通过HSP 70稳定19 S调节剂。26 S蛋白酶体的解离阻止Nrf 2降解，导致Nrf 2积累、磷酸化和核转位; 2）将HBE 1人细胞或原代人NHBE细胞暴露于轻度适应剂量的H2 O2，导致Nrf 2与20 S蛋白酶体亚基基因和Pa 28 α β γ基因的ARE（或EpRE）序列结合，并在H2 O2暴露后5-10小时内上调其表达。Irf-1和/或NFκB转录因子同时与三种免疫蛋白酶体基因的上游区域结合，上调免疫蛋白酶体和Pa 28 α β的表达; elegans和D.黑腹果蝇对H_2O_2的反应需要20 S蛋白酶体和Pa_（28）γ的表达，通过Nrf 2的skn-1和cnc-C同源物进行; 4）在衰老的HBE 1和原代NHBE细胞中，在我们的非分裂HBE 1和NHBE细胞的高氧加速老化模型中，以及在来自老年人供体的NHBE细胞中，适应氧化应激的能力下降，部分原因是蛋白酶体的Nrf 2和Irf-1/NFκB信号传导减少，Pa 2 α-γ和免疫蛋白酶体合成。H2 O2通过Nrf 2同源物skn-1和cnc-C诱导的Pa 28 γ和蛋白酶体的表达也随着C. elegans和D.黑腹果蝇，导致与年龄相关的整体抗应激能力和适应性下降。

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 Pa28αßγ genes, and up-regulate their expression within 5-10 hours after H2O2 exposure. The Irf-1 and/or NFκB transcription factors simultaneously bind to upstream regions of the three Immunoproteasome genes, and up-regulate Immunoproteasome and Pa28αß expression; 3) Adaptation of C. elegans and D. melanogaster to H2O2 requires 20S Proteasome and Pa28γ 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/NFκB signaling of Proteasome, Pa2αßγ, and Immunoproteasome synthesis. H2O2-induced expression of Pa28γ 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.