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.
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