Nitric Oxide Mediated Degradation of CYP2A6 via the Ubiquitin-Proteasome Pathway in Human Hepatoma Cells Public
Cerrone, John (Spring 2020)
Published
Abstract
Several cytochrome P450 enzymes are known to be down regulated by nitric oxide (NO). CYP2A6 is responsible for the metabolism of nicotine and several other xenobiotics, but its susceptibility to down-regulation by NO has not been reported. To address this question, we used HuH7 human hepatoma cell lines to express CYP2A6 with a C-terminal V5 tag (CYP2A6V5). NO donor treatment (DPTA NONOate, DPTA), downregulated CYP2A6 protein to approximately 40% of control levels in four hours. An NO scavenging agent protected CYP2A6 from down- regulation by DPTA in a concentration-dependent manner, demonstrating that the down- regulation is NO-dependent. Experiments with the protein synthesis inhibitor cycloheximide showed that CYP2A6 protein down-regulation occurs post-translationally. In the presence of proteasome inhibitors MG132 or bortezomib, NO treated cells showed an accumulation of a high molecular mass signal, whereas autophagy inhibitors chloroquine and 3-methyladenine and the lysosomal and calpain inhibitor E64d had no effect. Immunoprecipitation of CYP2A6 followed by Western blotting with an anti-ubiquitin antibody showed that the high molecular mass species contain polyubiquitinated CYP2A6 protein. This suggests that NO led to the degradation of protein via the ubiquitin-proteasome pathway. The down-regulation by NO was blocked by the reversible CYP2A6 inhibitor pilocarpine but not by the suicide inhibitor methoxsalen, demonstrating that down-regulation requires NO access to the active site but does not require catalytic activity of the enzyme. These findings provide novel insights towards the regulation of CYP2A6 in a human cell line and can influence our understanding of 2A6 related drug metabolism.
Table of Contents
Table of Contents
1. INTRODUCTION 1
1.1 Nitric Oxide & Enzymatic Regulation 1
1.2 Cytochrome P450s & Regulation by Nitric Oxide 2
1.3 Proposal 3
2. MATERIALS AND METHODS 4
2.1 Materials and Reagents 4
2.2 Cell Culture 5
2.3 SDS-PAGE and Western Blot Assay 6
2.4 CYP2A6 Activity Assay 6
2.5 Ubiquitination Assay 7
2.6 Data Analyses 7
3. RESULTS AND DISCUSSION 8
3.1 Down-regulation of CYP2A6 by NO Donors 8
3.2 Effect of Protein Synthesis Inhibitor Cycloheximide 10
3.3 Attenuation of Down-regulation by PTIO 11
3.4 Effects of Protease Inhibitors on down-regulation of CYP2A6 by NO 14
3.5 Enhancement of High Molecular Mass Species by NO Donor & Proteasome Inhibitor 16 Treatments
3.6 Ubiquitination of CYP2A6 18
3.7 Effect of CYP2A6 Inhibitors on its NO-dependent degradation 19
3.8 Visualization of bound Methoxsalen and Pilocarpine 23
4. CONCLUSION 24
5. REFERENCES 28
6. FUNDING ACKNOWLEDGEMENTS 34
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