Many environmental toxicants are oxidants. Proteins are major targets of oxidative modification. They lose function & structure, and must be proteolyticaly degraded or they will aggregate and form cross-linked cellular inclusion bodies. In previous grant cycles, 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 both 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 WI-38 or HBE1 cells to hydrogen peroxide (H2O2), causes transient and reversible (< 5hr) 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 by PKC?, and nuclear translocation mediated by Akt; 2) Exposure of WI-38 or HBE1 cells to an adaptive (nM to low ?M) dose of H2O2, causes Nrf2 to bind to ARE/EpRE sequences of 20S Proteasome subunit genes and Pa28 genes and up-regulate expression of their protein products 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 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 WI-38 cells and in our model of hyperoxia-accelerated aging of non- dividing WI-38 and HBE1 cells, partly due to diminished Nrf2 and Irf-1/NF?B signaling of Proteasome, and Immunoproteasome synthesis. H2O2-induced expression of Pa28 and Proteasome, through the Nrf2 homologs skn-1 and cnc-C, also decays during aging of C. elegans and D. melanogaster, contributing to an age-related decline in overall stress resistance and adaptability.
Oxidative damage to proteins is a well-characterized outcome of exposure to a wide variety of environmental toxicants. So serious is the problem of environmental oxidants that the NIEHS made discovering the mechanisms of oxidative stress a major focus in its 2006-2011 Strategic Plan, entitled New Frontiers in Environmental Sciences and Human Health. We seek to identify the mechanisms by which the proteasome enzyme protects against environmental oxidative stress, and allows us to adapt to increased stress levels.
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