Volume 8 Issue 10 - May 22, 2009
Pregnane X receptor polymorphism affects CYP3A4 induction via a ligand-dependent interaction with steroid receptor coactivator-1
Jin-ding Huang

Institute of Biopharmaceutical Sciences, College of Medicine, National Cheng Kung University

Pharmacogenetics and genomics 17: 369-382 (2007)

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Drugs are metabolized by cytochrome P450, a large enzyme family.  The most important Cytochrome P450 in human is CYP3A4.  The metabolism by CYP3A4 increases along the use of some drugs, for example refampicin.  This is called drug induction.  Drug induction seems to be an important defense mechanism of human body, but causes therapeutic failure in disease.

The nuclear receptor PXR is an important mediator of CYP3A4 induction.  In the figure below, you can see xenobiotics (▲) or ligands binding to nuclear receptor.  The binding complex together with another nuclear receptor RXR sits on CYP3A4 promoter and turns on CYP3A4 expression.  In this way, refampicin can bind PXR and increase CYP3A4 enzyme.  More CYP3A4 will then metabolize more refampicin.  Refampicin therefore lose its action to kill TB bacteria.  A lot of other drugs metabolized CYP3A4 also lose its activity by coadministration with refampicin. The induction is a serious problem in drug therapy.

A few years ago, we found a genetic polymorphism of PXR (Y. P. Lim et al. Pharmacogenet. Genom. 15, 337-341 (2005)).  The Q158K mutation of PXR removes the drug induction by refampicin.  The mutation has a clear clinical significance.  We however did not understand why the amino acid Q158 of PXR is so important.  According to the crystal structure of PXR, Q158 position is located at α1 helix, outside the ligand binding site.  The mutation does not affect refampicin binding or PXR amount.  We proposed a hypothesis that the mutation affects protein-protein interaction of PXR and other proteins.

As shown below, activation or repression of a gene needs a lot of transcriptional factors.  We studied the literature and decided to work on SRC-1 and SMRT.

One of the most popular ways to study a protein-protein interaction is mammalian two-hybrid method.  As shown in the figure below, the method uses two proteins, GAL4 and VP16.  GAL4 is a DNA binding domain and VP16 is activation domain.  We used vector pBIND to fuse SRC-1 and GAL4 and vector pACT to fuse PXR(Q158) or PXR(K158) with VP16.  If SRC-1 can bind PXR, the binding will bring GAL4 and VP16 together and turn on the luciferase reporter gene.  The chemiluminescence intensity will tell the protein-protein interaction.

The last figure shows one of our experimental results.  Q158-PXR binds with SRC-1, but not K158-PXR.  It is clear that amino acid Q158 of PXR is critical in the interaction of PXR and SRC-1.  This is actually the first illustration how the genetic polymorphism affects protein-protein interaction at the transcriptional level.

In addition to Q158K mutation, we also created 9 other mutations of PXR at different locations.  We also tested three other drugs, clotrimatrole, paclitaxel, and nifedipine, in addition to refampicin.  When different mutants and different ligands are used, it was found that SRC-1 and PXR interaction is ligand-dependent.  Interestingly, PXR with different ligands interact with SRC-1 differently.

On the other hand, we did not find the interaction of PXR and the repressor SMRT.  The mutation of Q158K did not affect PXR-SMRT interaction much.  The interaction of PXR and SMRT is also not much dependent on ligands when different mutant PXR are used.  At the silent stage, PXR interact with SMRT.  When ligands arrive, liganded-PXR leaves SMRT and sits on CYP3A4 promoter with RXR.  Following the event, SRC-1 brings other proteins such as CBP/p300 to PXR/RXR.  The gene transcription is then actively going on.

1. Y. P. Lim, C. H. Liu, L. J. Shyu, J. D. Huang (2005) Functional characterization of a novel polymorphism of pregnane X receptor, Q158K, in Chinese subjects. Pharmacogenet. Genom. 15, 337-341.
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