Cytochromes P450 3A are ER membrane-anchored hemoproteins that are predominantly responsible for hepatic metabolism of endo- and xenobiotics. Hence mechanisms regulating hepatic CYP3A content can significantly influence drug metabolism, therapeutics and clinical drug-drug interactions. Our goal therefore is to characterize the molecular mechanisms involved in the stabilization and turnover of CYP3A. In the rat/human liver, native and suicidally inactivated CYPs 3A are degraded via ubiquitin-dependent 26S proteasomal degradation. The proteasomal targeting of CYP3A4 requires phosphorylation at residues S478, S420 and T264 (based on in-vitro data and S35-pulse-chase analyses of wild type and mutant CYPs 3A expressed in HepG2 cells), followed by ubiquitination by the mammalian ubiquitin ligase complexes, and its subsequent p97 AAA ATPase-dependent ER-extraction into the cytosol. However, given that the CYP3A bulk is in the cytosol and a significant 26S proteasomal fraction is ER-associated, it was plausible that the 26S proteasomal AAA ATPases (particularly the Rpt4 19S cap subunit which has been shown to extract ER-membrane proteins into the cytosol) were also involved in CYP3A extraction. To determine the relative roles of p97 and 26S proteasome, we used lentiviral shRNA-mediated knockdown of rat hepatic p97 and proteasomal Rpt4 subunit in combination with MG132 (a proteasomal inhibitor) or hemin (a reported proteasomal AAA ATPase inhibitor) as probes. Knockdown of p97 or Rpt4 did not impair 20S proteasomal proteolytic core function. However, p97 knockdown (90%) nearly completely abolished the membrane extraction of CYP3A, resulting in a marked accumulation of functionally active parent CYP3A and its ubiquitinated species that remained firmly ER-tethered. We document that CYP3A ubiquitination precedes its membrane extraction. Upon p97 knockdown, little CYP3A was extracted into the cytosol, even after MG262-proteasomal inhibition. Hemin blocked p97 AAA ATPase activity and consistent with this inhibition both parent and ubiquitinated CYP3A species accumulated in the ER. Thus, p97 plays a major and obligatory role in the ER extraction of CYP3A. By contrast following 92% of Rpt4 knockdown, S35-CYP3A pulse chase analyses revealed that CYP3A extraction into the cytosol was unhindered. However, once CYP3A was extracted into the cytosol it was modestly stabilized. These findings are consistent with mutually exclusive albeit sequential roles for p97 and Rpt4 AAA ATPases in CYP3A ERAD, wherein Rpt4 functions at the proteasomal degradation step following ER extraction of CYP3A by p97. These studies elucidate key steps in the mechanism of regulation of liver CYP3A turnover via ERAD.
Supported by NIH grants DK26506 and GM44037.