In vivo and in vitro degradation of cytochrome P-450 2E1: A potential role for ubiquitin-mediated proteolysis

 
 
 
 

In vivo and in vitro degradation of cytochrome P-450 2E1: A potential role for ubiquitin-mediated proteolysis

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Title: In vivo and in vitro degradation of cytochrome P-450 2E1: A potential role for ubiquitin-mediated proteolysis
Author: Tierney, Daniel
Description: The heme protein cytochrome P450 2E1 is short-lived relative to other P450's suggesting regulation by a specific proteolytic mechanism. The ATP/ubiquitin-mediated degradation of P450 2E1 was investigated since ubiquitin functions in selective degradation of abnormal and short-lived proteins. Labilization of P450 2E1 by heme and/or protein modification or by removal of a stabilizing ligand could produce a conformationally labile form of P450 2E1 with enhanced susceptibility to degradation. P450 2E1 was induced in mice by an intragastric dose of acetone followed by the i.p. administration of the labilizer, carbon tetrachloride (CCl4) which promotes heme alkylation of P450 2E1, 1-aminobenzotriazole (ABT) which N-alkylates the heme prosthetic group, or 3-amino-1,2,4-triazole (3AT) which inactivates P450 2E1 without heme modification; or stabilizer, 4-methylpyrazole (4MP). Within 1 h of administration, P450 2E1 was inactivated by CCl4 or 3AT and approximately 80% of the induced enzyme was degraded; a significant loss of ABT-inactivated P450 2E1, however, was not observed. Degradation of P450 2E1 was accompanied by the formation of high molecular weight microsomal ubiquitin conjugates. P450 2E1 loss and conjugate formation loss was prevented by 4MP. T he ATP/ubiquitin-mediated degradation of P450 2E1 was investigated in vitro using rabbit reticulocyte Fraction II as a protease source. Microsomal or purified P450 2E1 were inactivated with the labilizers described above. CCl4-inactivated P450 2E1 was degraded in the absence or presence of ATP and ubiquitin; degradation of 3AT-inactivated P450 2E1 was ATP and ubiquitin dependent; and ABT-inactivated P450 2E1 was not degraded by Fraction II. Hemin or the ubiquitin mutant, Ub-C48, (specific inhibitors of ubiquitin-mediated degradation) effected partial or complete inhibition of CCl4- or 3AT-inactivated P450 2E1, respectively. 4MP prevented the ATP-independent degradation of the enzyme. P450 2E1 degradation under all conditions was dependent on NADPH. These studies indicate that differentially modified forms of P450 2E1 possess characteristic susceptibilities to degradation; only certain forms appear appropriate for ATP/ubiquitin-mediated degradation
Permanent Link: http://rave.ohiolink.edu/etdc/view?acc_num=case1059667227
http://hdl.handle.net/2374.OX/16756
Date: 1992

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