CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases.
Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae, it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established.
Pabarcus
Michael K
MK
Department of Cellular and Molecular Pharmacology, University of California San Francisco, 94158-2280, United States.
Hoe
Nicholas
N
Sadeghi
Sheila
S
Patterson
Cam
C
Wiertz
Emmanuel
E
Correia
Maria Almira
MA
D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D008565:Membrane Proteins; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C555000:AMFR protein, human; C504816:MARCHF6 protein, human; D060285:Receptors, Autocrine Motility Factor; C467728:STUB1 protein, human; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases