Liver cytochrome P450 3A endoplasmic reticulum-associated degradation : a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol
The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2011 |
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| Erschienen: |
2011 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:286 |
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| Enthalten in: |
The Journal of biological chemistry - 286(2011), 5 vom: 04. Feb., Seite 3815-28 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Acharya, Poulomi [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 21.03.2011 Date Revised 20.10.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1074/jbc.M110.186981 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM203879244 |
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| 100 | 1 | |a Acharya, Poulomi |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Liver cytochrome P450 3A endoplasmic reticulum-associated degradation |b a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol |
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| 500 | |a Date Revised 20.10.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function | ||
| 650 | 4 | |a Journal Article | |
| 650 | 7 | |a Nuclear Proteins |2 NLM | |
| 650 | 7 | |a RNA, Small Interfering |2 NLM | |
| 650 | 7 | |a Cytochrome P-450 CYP3A |2 NLM | |
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| 650 | 7 | |a Adenosine Triphosphatases |2 NLM | |
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| 650 | 7 | |a p97 ATPase |2 NLM | |
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| 700 | 1 | |a Liao, Mingxiang |e verfasserin |4 aut | |
| 700 | 1 | |a Engel, Juan C |e verfasserin |4 aut | |
| 700 | 1 | |a Correia, Maria Almira |e verfasserin |4 aut | |
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