Cytochrome P450 Degradation and Its Clinical Relevance

Human Liver Cytochrome P450 3A4 Ubiquitination

“…Purified recombinant CYP3A4(His) 6 protein (250 pmol) was inactivated at 37°C for 15 min with CuOOH (1 mM), EDTA (1 mM), and GSH (1 mM) in 50 mM Hepes buffer, pH 7.4, containing 10% glycerol in a final volume of 30 l. DTT (2 mM, final) was added to quench the reaction. A similarly reconstituted system incubated in the absence of CuOOH served as the parallel native CYP3A4 control.…”

“…Conversely in system 5, UbcH5a-CHIP and gp78 were present from the start, but gp78-mediated ubiquitination was initiated only at 30 min by inclusion of its cognate E2 UBC7. The dual UBC7-gp78 and UbcH5a-CHIP-reconstituted ubiquitination system (system 3) consisted of UBA1 (E1, 0.1 M), human UBC7 (2 M), gp78C (1 M), UbcH5a (2 M), CHIP(His) 6 (1 M), Hsc70 (1 M), Hsp40 (1 M), HA-Ub (20 M), and CuOOH-inactivated CYP3A4 (4 M) and an ATP-regenerating system in Hepes buffer (50 mM, pH 7.4, containing 20% glycerol), EGTA (0.5 mM), EDTA (0.5 mM), with/without addition of PKC (0.016 units), and PKA (0.016 units) in a final volume of 120 l, and it was incubated at 30°C. Aliquots (30 l) of the reaction mixture were collected at 30, 60, and 90 min, mixed with 10 l of 4ϫ loading buffer, and boiled for 5 min.…”

“…To determine the role of the negatively charged surface clusters in CYP3A4 ubiquitination, selective patches consisting of three, four, or five amino acids were mutated to Ala by QuikChange Lightning mutagenesis kit (Agilent Technologies, Santa Clara, CA) or by customized order of cDNA synthesis by GenScript USA, Inc. (Piscataway, NJ), and subcloned into pTOPO3A4(His) 6 . The mutated CYP3A4(His) 6 fragments were excised from pTOPO vector using NdeI-KpnI double restriction enzyme digestion and then ligated into the pCWori ϩ vector for heterologous expression in Escherichia coli DH5␣F strain.…”

“…The mutated CYP3A4(His) 6 fragments were excised from pTOPO vector using NdeI-KpnI double restriction enzyme digestion and then ligated into the pCWori ϩ vector for heterologous expression in Escherichia coli DH5␣F strain. The primers and templates used in this mutagenesis and the Genscript orders for each construct are listed (Table 1).…”

“…Of these, CYP3A4 is not only the most abundant P450 in the human liver, it is also the most dominant catalyst, responsible for the metabolism and elimination of over 50% of clinically relevant drugs (1,2). Alteration of its functional hepatic content either through drug-mediated induction (enhanced expression) or inhibition and/or inactivation and subsequent proteolytic degradation is associated with serious therapeutic consequences due to altered pharmacological drug responses and/or drug-drug interactions (1)(2)(3)(4)(5)(6)(7). We have documented that the proteolytic degradation of the native structurally and functionally intact CYP3A4 and/or its rat or mouse orthologs (CYPs 3A), and their suicidally inactivated counterparts occurs via the ubiquitin (Ub)-mediated 26 S proteasomal system (UPS) (8 -12).…”