Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.
We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.
Zangar
Richard C
RC
Pacific Northwest National Laboratory, 902 Battelle Blvd., MS P7-56, Richland, WA 99352, USA. richard.zangar@pnl.gov
Kocarek
Thomas A
TA
Shen
Shang
S
Bollinger
Nikki
N
Dahn
Michael S
MS
Lee
Donna W
DW
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D020868:Gene Silencing / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D051381:Rats
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde