title: Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.
abstract: CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation.
authors.affiliation: From the Departments of Cellular and Molecular Pharmacology.
authors.forename: YongQiang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the Departments of Cellular and Molecular Pharmacology.
authors.forename: Sung-Mi
authors.initials: SM
authors.lastname: Kim
authors.affiliation: Pharmaceutical Chemistry, and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Trnka
authors.affiliation: From the Departments of Cellular and Molecular Pharmacology.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Pharmaceutical Chemistry, and.
authors.forename: A L
authors.initials: AL
authors.lastname: Burlingame
authors.affiliation: From the Departments of Cellular and Molecular Pharmacology, Pharmaceutical Chemistry, and Bioengineering and Therapeutic Sciences, The Liver Center, University of California at San Francisco, San Francisco, California 94158-2517 almira.correia@ucsf.edu.
authors.forename: Maria Almira
authors.initials: MA
authors.lastname: Correia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D060285:Receptors, Autocrine Motility Factor / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases
keywords: CHIP; CYP3A4; Cytochrome P450; E3 Ubiquitin Ligase; Endoplasmic Reticulum-associated Protein Degradation (ERAD); Mass Spectrometry (MS); Protein Phosphorylation; Ubiquitin-conjugating Enzyme (E2 enzyme); Ubiquitylation (ubiquitination); gp78/AMFR