Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum

Abstract

To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase , which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention "receptor" since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

“…The evidence presented supports a biphasic effect on CYP3A levels monitored by immunoblotting and confocal immunofluorescence analyses. It confirms previous reports (Wang et al, 1999;Faouzi et al, 2007;Szczesna-Skorupa and Kemper, 2008) by clearly documenting that at the lower concentrations, MG132 indeed stabilizes CYP3A content, whether monitored by immunoblotting, immunofluorescence, or pulse-chase coupled with CYP3A immunoprecipitation analyses. However, pulse-chase analyses of CYP3A…”

“…9), CYP3A was also found to exhibit perinuclear accumulation and plasma membrane migration after 18 h of MG132 treatment. This ER to plasma membrane migration was apparently dependent on nocodazole-sensitive microtubular network (Szczesna-Skorupa and Kemper, 2008). 3 The cause for this MG132-elicited cellular P450 relocalization is unclear.…”