We are constantly exposed to toxic chemicals that require metabolic activation to products responsible for cell injury. Cytochrome P450 2E1 has an important role in human health as a result of being induced by acute and chronic alcohol ingestion, catalyzing the bioactivation of many established carcinogens and hepatotoxins, and has been implicated as a causative factor in alcoholic liver disease. The activity of P450 2E1 depends on its concentration that is regulated by both the rate of synthesis and the rate of degradation. Studies have shown the importance of the degradative path in the regulation of P450 2E1 in adult liver. Despite this fact, little is known about the mechanism of enzyme loss. Our working hypothesis is that P450 2E1 undergoes degradation by both lysosomal and nonlysosomal pathways. the selectivity of the pathway is dictated by changes that occur in the structure of P450 2E1 as a result of labilization and these structural changes are recognized by cytosolic proteolytic systems. Cytosolic proteolysis represents the rapid degradation of the enzyme and its inhibition results in induction of the enzyme. The overall goal of this proposal is to characterize the pathways for P450 2E1 degradation and to determine how ethanol affects them. We are proposing a systematic investigation of the degradation of P450 2E1 using studies in vitro with microsomal suspensions and purified P450 2E1, studies in cells in culture with expressed P450 2E1, and studies in vivo after the induction of P450 2E1 by an acute dose of acetone as well as long term ethanol treatment. The proposal has four specific aims. 1. Characterize the damage that occurs during the labilization of P450 2E1 with CCI4, 3- amino-1,2,4-triazole (3AT), diethydithiocarbamate (DEDTC), and H2O2. This will be done with purified P450 2E1 and subsequent isolation of the modified protein and or peptides by HPLC and sequence analysis of modified proteins. We will look for heme-linked peptides and oxidatively damaged amino acids. 2. Characterize the degradation of P450 2E1 in vitro using purified components of the ubiquitin-dependent and the multicatalytic proteinase (MCP) pathways. Proteolysis with purified components of the ubiquitin-dependent and MCP pathways will be used in vitro with differentially modified forms of P450 2E1. This includes suicide inactivation, phosphorylation with protein kinase A (PKA), protein kinase C (PKC) and Ca+2-calmodulin dependent protein kinase (CaM kinase II), and enzyme modified by site directed mutagenesis and expressed E. coli. 3. Characterize the degradation of P450 2E1 after stable expression in Chinese Hamster Ovary (CHO-K1) cells. The full length P450 2E1 cDNA will be subcloned into expression vectors and transfected into CHO-K1 cells in culture. The half-life of the enzyme will be determined by pulse-chase experiments. Questions concerning the degradative pathway will be tested by using specific inhibitors and site-specific mutations of the enzyme. 4. Characterize the degradation of P450 2E1 in mice after an acute dose of acetone and in rats after chronic ethanol by enteral feeding. Affects of these treatments on the proteolytic systems identified in Aims 1, 2 and 3 will be determined. Together, the results from these studies will provide important information about the affect of ethanol on P450 2E1 degradation as well as the degradation of other proteins after ethanol treatment.
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