OXYGEN RADICAL TOXICITY AND PROTEIN DEGRADATION

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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.