CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases

Abstract

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.

Fig. 1

Functional competence of the reconstituted UBC7-dependent ubiquitination systems. The various combinations of E2s (UBC7 or UbcH5a) and E3s were tested for competence in forming Ub-dimers and/or polyUb chains in the presence of E1 and ³²P-Ub. Component dependence is shown for ATP, UBC7 or UbcH5a, and the various E3s. UbcH5a/CHIP reactions also included Hsp70, Hsc70 and Hsp40. The reactions contained E1 (0.1 μM), UBC7 (4 μM), ³²P-Ub (20 μM), ATP (4 mM), CPK (20 U/ml), CP (20 mM), MgCl₂ (10 mM), DTT (1 mM), and one of the following E3s: GST-gp78C (1 μM), GST-HRD1CT1A (10 μM), GST-HRD1CT1B (10 μM), GST-TEB4r (10 μM), or its inactive mutant GST-TEB4rC9A (10 μM). UbcH5a and His-CHIP-dependent ubiquitination reactions contained: UbcH5a (1.5 μM) instead of UBC7, His-CHIP (4 μM) as the E3, Hsc70 (2.5 μM), and Hsp40 (2.5 μM), in an otherwise identical reaction mixture. The mixtures were incubated at 30 °C for 90 min and analyzed as detailed in Experimental Procedures. Data from a typical experiment are shown. Each experiment was conducted in its entirety at the least 3 separate times.

Fig. 2

CYP3A4 reconstituted ubiquitination reactions. Each ubiquitination system identical to the complete system described in Fig. 1 was tested with and without CuOOH-inactivated His3A4 (250 pmol) in the presence of each E3’s cognate E2 at concentrations indicated in Fig. 1. E3 and ATP dependence are shown for all systems. Color contrasted images were generated using ImageQuant to enhance visualization of polyubiquitination effects. For further details see Experimental Procedures. Data from a typical experiment are shown. Each experiment was conducted in its entirety at the least 3 separate times. The color wheel intensity code for these PhosphorImager data is as follows: Black>dark blue>light blue>green>yellow>orange>red>magenta>white. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Immunoprecipitation/immunoblotting analyses of UBC7/gp78C-catalyzed CYP3A4 ubiquitination. Each UBC7/gp78C ubiquitination system identical to the complete system described in Fig. 1 except for the presence of ³²P-Ub (167 μM), was incubated at 30 °C for 90 min with or without CYP3A4 (250 pmol) in the presence or absence of ATP exactly as detailed (Experimental Procedures). An aliquot of each incubation was subjected to SDS–PAGE with PhosphorImager analyses (A). Other aliquots were subjected to immunoprecipitation analyses with goat anti-CYP3A4 IgGs as detailed (Experimental Procedures). Aliquots of these immunoprecipitates were then subjected to SDS-PAGE with PhosphorImager analyses (B) or immunoblotting analyses with anti-Ub IgGs as the primary antibody, followed by alkaline phosphatase as the secondary (C) as detailed [9]. A direct comparison of immunoprecipitates of aliquots of reactions carried out in the presence of Ub 20 μM (Fig. 2A) or 167 μM (Fig. 3A) are also shown (D). Data from a typical experiment are shown. Each experiment was conducted in its entirety at the least 3 separate times. The color wheel intensity code for these PhosphorImager data is as follows: Black>dark blue>light blue>green>yellow>orange>red>magenta>white. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Immunoprecipitation/immunoblotting analyses of the UbcH5a/CHIP-catalyzed CYP3A4 ubiquitination. Each UbcH5a/CHIP ubiquitination system identical to the complete system described in Fig. 1 except for the presence of ³²P-Ub (167 μM), was incubated at 30 °C for 90 min with or without CYP3A4 (250 pmol) in the presence or absence of ATP exactly as detailed (Experimental Procedures). An aliquot of each incubation was subjected to SDS–PAGE with PhosphorImager analyses (A). Another aliquot was subjected to immunoprecipitation analyses with goat anti-CYP3A4 IgGs as detailed (Experimental Procedures). Aliquots of these immunoprecipitates were then subjected to SDS–PAGE with PhosphorImager analyses (B). Data from a typical experiment are shown. The intense bluish green bands at the very top of either SDS–PAGE gel most likely reflect ubiquitinated CHIP and/or Hsp70, as they persist in the absence of CYP3A4 but not in the absence of ATP. Each experiment was conducted in its entirety at the least 3 separate times. The color wheel intensity code for these PhosphorImager data is as follows: Black>dark blue>light blue>green>yellow>orange>red>magenta>white. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

CYP3A4 ubiquitination by UBC7/gp78 and UbcH5a/CHIP systems: Plausible scenarios for physiological redundancy or functional complementarity. Following structural inactivation in vivo, ER-bound CYP3A4 could be ubiquitinated by (1) UBC7/gp78 and/or (2) UbcH5a/CHIP. The ER-polytopic gp78-RING E3 ligase catalyses polyUb chain assembly on UBC7 before transferring the elaborated polyUb chain onto accessible CYP3A4 K-residue(s). The ubiquitinated CYP3A4 is then extracted out of the ER-membrane by the AAA ATPase p97 complex and delivered to the 26S proteasome for destruction in a classical ERAD process. Alternatively or concomitantly, exposure of normally buried hydrophobic sequences within the CYP3A4 active site following structural inactivation, could target it to the cytosolic Hsp70/Hsc70/Hsp40 molecular chaperone complex. Such Hsp70-corralled CYP3A4 becomes recognized by the cytosolic CHIP E3 ligase-tetratricopeptide repeat (TPR) motif, resulting in its subsequent ubiquitination by the UbcH5a/CHIP E3 Ub-ligase U-box domain. The ubiquitinated CYP3A4 could then be similarly delivered by the AAA ATPase p97-complex to the 26S proteasome for destruction. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)