Characterizing the Downregulation of CYP2J2 by Nitric Oxide Open Access
Cheng, Joan S. (2017)
Published
Abstract
Arachidonic acid (AA), antihistamine drugs, anticancer drugs, and other relevant drugs are metabolized by the cytochrome P450 enzyme CYP2J2. Its metabolism of AA is especially important, as the metabolites formed can protect against inflammation and hypertension but also promote carcinoma metastasis. Thus understanding how the enzyme is regulated within the cell, particularly by nitric oxide (NO), is pertinent.
In the human body, NO is produced in small amounts by three main isoforms of nitric oxide synthase. Overproduction has been linked to several disease states, and NO itself is a highly reactive molecule capable of permanently modifying macromolecules and inducing cell apoptosis. As a result, NO was thought to lack the specificity to participate in cell signaling events. However, evidence now points to the role of NO in signaling and regulation, particularly of P450 enzymes.
Experiments with Huh7-2J2v5, a human liver cancer cell line stably transduced with CYP2J2v5, reveal that NO donated from dipropylenetriamine NONOate (DPTA) downregulated CYP2J2 in a time and concentration-dependent manner. Since CYP2J2 mRNA levels were unaffected by NO, and the translational inhibitor cycloheximide was unable to block downregulation, it was concluded that protein degradation was occurring. Although preliminary data showed that the proteasomal inhibitor bortezomib was able to partially restore CYP2J2 levels, suggesting an ubiquitination-dependent pathway, high molecular weight (ubiquitinated) species were not observed. Furthermore, additional experiments concluded that bortezomib as well as the autophagy inhibitors 3-methylamine and chloroquine were not actually able to block downregulation. Thus downregulation occurs through neither an ubiquitin-dependent nor lysosomal pathway.
Once the mechanism is fully characterized by further studies using other protein degradation inhibitors, it will be tested to see whether it occurs in cardiac myocytes, where CYP2J2 is expressed a naturally higher levels. Overall, these findings are significant to understanding how to treat various disease states related to abnormal CYP2J2 and/or NO expression.
Table of Contents
Table of Contents
ABSTRACT
iv
ACKNOWLEDGMENTS
vi
1. INTRODUCTION
1.1. Cytochrome P450 and CYP2J2
1.2. Nitric Oxide as a Regulator
1.3. Proposal
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2. MATERIALS AND METHODS
2.1. Materials and Reagents
2.2. Cell Culture
2.3 Protein Extraction and Western Blot
2.4. Reverse Transcriptase and Real-Time qPCR
2.5. Transfection and Immunoprecipitation
2.6. Data Analysis and Statistics
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3. RESULTS AND DISCUSSION
3.1 Effect of DPTA on CYP2J2 Protein Expression
3.2. Effect of DPTA on CYP2J2 mRNA Expression
3.3. Effect of DPTA on CYP2J2 Protein Expression for Huh7-2J2v5 Cells Treated With CHX
3.4. Detection of High Molecular Weight Species through Immunoprecipitation
3.5. Effect of DPTA on CYP2J2 Protein Expression of Huh7-2J2v5 Cells Treated With Protein Degradation Inhibitors
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4. CONCLUSION
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5. REFERENCES
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