Abstracts F1-F4

Category F. Drug Metabolism, Toxicity and Pharmacogenomics

 

F1.  Pregnane X receptor SUMOylation and De-SUMOylation

Chang Liu, Wenqi Cui and Jeff L. Staudinger
Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, Kansas, USA

Patients undergoing pharmacotherapy with certain medications can exhibit a compromised immune response in liver and intestine.  Previous results from our laboratory indicate that modification of pregnane X receptor (PXR, NR1I2), a member of the nuclear receptor superfamily of transcription factors, by small ubiquitin-related modifier (SUMO) contributes to this phenomenon (Hu et al., 2010).  Here, we further examine PXR SUMOylation/de-SUMOylation reaction mechanisms using in vitro and cell-based methods.  Bacterial expression and purification systems were used in conjunction with in vitro SUMOylation reactions to further analyze PXR SUMOylation by both SUMO1 and SUMO3.  The E3 SUMO-protein ligase protein inhibitor of activated STAT protein 4 (PIAS4) potentiates SUMOylation of the PXR ligand binding domain (PXR-LBD) in vitro.  Using cell-based methods we characterize the de-SUMOylation of PXR using six different sentrin protease (SENP) enzymes including SENP1, SENP2, SENP3, SENP5, SENP6, and SENP7.  Both SENP1 and SENP2 effectively de-conjugate either SUMO1 or SUMO3 from PXR, whereas SENP6 inhibits the formation of SUMO3 chains.  Collectively, our data suggest that (1) PXR can serve as a substrate for either SUMO1 or SUMO3 in vitro and in cell-based assays, (2) both SENP1 as well as SENP2 are able to remove SUMO moieties in a cellular environment, and (3) that SENP3 and SENP6 remove or prevent the formation of SUMO-chains on PXR.  Future efforts should seek to determine the extent to which the biochemical mechanisms described here function in human liver and intestine undergoing therapy with PXR activators.

-top-

 

F2.  Biochemical Characterization of Pregnane X Receptor Ubiquitination and SUMOylation

Mengxi Sun and Jeff L. Staudinger
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas

Highly expressed in the entero-hepatic system, the nuclear receptor pregnane X receptor (PXR, NR1I2) is a well-characterized ‘xenobiotic sensor’ that can be activated by a wide array of xenobiotics and prescription drugs.  The net effect of PXR activation is to increase metabolism and clearance of drugs and xenobiotics from the body by transactivating genes that encode drug processing enzymes.  The complete understanding of PXR biology is thus important for the prevention of adverse drug-drug interactions and the development of safe and effective pharmacotherapies.  Recent research recognizes that post-translational modification (PTM) of PXR (e.g; ubiquitination and SUMOylation) significantly impacts PXR biological function; however, these studies are still in their infancy. Here we present a biochemical study of the regulated ubiquitination and SUMOylation of the PXR protein.  Our data indicate that PXR is a target of both ubiquitin and SUMO signaling pathways.  These two signal transduction pathways likely interface with ligand-activated PXR to modify its biological activity in specific disease states such as inflammation.  Future experiments will seek to employ a linear Ub-PXR/SUMO-PXR fusion protein approach to explore the biological significance of these post-translational modifications.

-top-

F3.  Biochemical Methods to Detect post-Translational Modification of Human Pregnane X Receptor

Wenqi Cui, Mengxi Sun, Sarah Woody, and Jeff L. Staudinger
Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, Kansas 66045, USA

Patients undergoing pharmacotherapy with the antibiotic rifampicin can exhibit compromised immune-responses in liver and intestine.   Pregnane x receptor (PXR, NR1I2), a member of the nuclear receptor superfamily of ligand-activated transcription factors, is strongly activated by rifampicin, as well as other drugs, in these tissues.  The current molecular paradigm suggests that inflammation-dependent SUMOylation of PXR on specific lysine (K, Lys) residue(s) plays a pivotal role in suppression of the immune-response.  There are 28 lysine residues in human PXR protein.   A systematic site-directed mutagenesis approach is used to reveal two major sites of SUMOylation at positions K108 and K128 on the PXR protein.  The current project is focused on defining the functional consequence of these mutant PXR proteins with respect to biological activity and trans-repression of inflammatory signaling pathways mediated by NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells).  Additional efforts are focused on developing a dual epitope-tagged PXR adenoviral expression vector for high level expression in cultured cells and subsequent two-step purification using a novel protocol.  The purified protein will be used in conjunction mass-spectrometry to identify additional ligand- and inflammation-induced post-translational modification(s) that target the human PXR protein.  Data from these studies is expected to produce novel strategies for high-throughput screening of drug candidates that could be used to suppress immune response in liver and colon a predicted way for patients experiencing inflammatory bowel and liver diseases, for which current treatments are largely ineffective.

-top-

 

F4.  SUMO-modification Alters PXR Transactivation

Sarah K. Woody, Mengxi Sun and Jeffrey L. Staudinger
University of Kansas, Department of Pharmacology and Toxicology, Lawrence, KS

Pregnane X Receptor (PXR, NR1I2) is a ligand-activated nuclear receptor superfamily member expressed at high levels within the entero-hepatic system in mammals. The primary function of PXR is to regulate the metabolism and transport of xenobiotics and endogenous compounds by controlling the expression of genes encoding major drug metabolizing enzymes and drug transporter proteins. To initiate transcription, PXR must interact with a multiple co-activator proteins including, but not limited to, Steroid Receptor Co-activator proteins (SRC-1/SRC-2/SRC-3) and p300/CBP co-integrator proteins.  These co-activator proteins facilitate the recruitment of transcriptional mediator complexes as well as RNA polymerase II and therefore initiate transcription.  SUMOylation is a reversible post-translational modification that plays a central role in regulating multiple cellular activities such as protein-protein interactions. While direct PXR-target gene activation programs are currently well characterized, little is known about the effect of SUMO-modification on PXR. Our working hypothesis is that SUMO-modified PXR alters its protein-protein interaction profile. Thus, we have created a GAL4-PXR fusion system that allows us to directly examine SUMO-PXR interaction with the aforementioned PXR-binding proteins. Future efforts are focused on developing novel methods for identification of SUMO-PXR-binding proteins using non-biased methods.

Methods and Materials

Plasmids.  In order to create GAL4-PXR fusion proteins, full-length human PXR (hPXR) was first PCR amplified utilizing two different sets of oligonucleotide primers (Table 1).

Table 1 – Oligonucleotide primer sequences

Protein

Primer Sequences

Enzymes used:

GAL4-hPXR (Full-length)

NO PCR NEEDED

  5’ EcoRI and 3’ BamHI

GAL4-hPXR DBD

  Left:   5’ - gAC gCC gAA TTC ATg gAg gTg AgA CCC AAA gAA AgC - 3’

  Right:   5’- gAC gCC gAA TTC TCA gCT CTC CAg gCA CTT gCg Cag - 3’

  5’ and 3’ - EcoRI

GAL4-hPXR LBD

  Left:   5’- gAC ggC gAA TTC ggC ATg AAg AAg gAg ATg ATC ATg - 3’

  Right:  5’- gAC ggC CTC gAg TCA gCT ACC TgT gAT gCC gAA C - 3’

  5’ EcoRI and 3’ Xho I

Following PCR, amplimers and full-length hPXR were subjected to restriction enzyme digestion utilizing the enzymes presented in Table 1.  Simultaneously, the pM vector (Clontech), containing the GAL4-DNA binding domain, was subjected to restriction enzyme digestion using either EcoRI/BamHI (Full-length), EcoRI (DBD), or EcoRI/Sal I (LBD).  Following digestion, DNA fragments were gel purified and precipitated.  Corresponding DNA fragments (vector and insert) were ligated utilizing T4 DNA Ligase (New England BioLabs) according to the manufacturer’s instructions.  Constructs were sequenced to ensure integrity of the DNA sequence followed by transfection and western blotting to confirm expression.

Western Blotting. CV-1 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.  Forty-eight hours post-transfection cells were lysed in 2X sodium dodecyl sulfate (SDS) with 50mM dithiothreitol (DTT).  Whole cell lysates were resolved via SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane.  Membranes were probed with a mouse monoclonal GAL4 antibody (Thermo Scientific) at a 1:1000 dilution followed by goat anti-mouse IgG-HRP (Santa Cruz) at 1:5000. Immunoreactivity was detected using Pierce ECL Western Blotting Substrate (Thermo Scientific).

Cell-based SUMOylation Assay. CV-1 cells were transfected with GAL4-hPXR constructs along with pcDNA4-His-tagged SUMO1 or SUMO3 and pCMV-Flag PIAS1 or PIASy.  Forty-eight hours post-transfections cells were lysed and subjected to a cell based SUMOylation assay as previously described2.  Protein samples were resolved via SDS-PAGE as previously described.  Immunoblot analysis was used to detect SUMOylated hPXR using mouse monoclonal PXR H-11 antibody (1:5000 dilution, Santa Cruz).

PXR Transactivation Assay.  CV-1 cells were plated on 96-well plates at a density of 7,000 cells/well as previously described1. Each well was transfected with a combination of 30 ng of the reporter gene pFR Luciferase, 30 ng of CMV-β-Gal, 10 ng GAL4-hPXR construct, 10 ng of full length co-factors, 10 ng of SUMO1 or SUMO3 and 10 ng of the SUMO-E3 ligases, PIAS1 or PIASy.  pBluescript was added as needed to bring the final DNA concentration to 110 ng/well.  Twenty-four hours post-transfection, cells were treated with DMSO or 10 μM Rifampicin (Rif) for 18 hours.  Luciferase activity was determined using the Luciferase Assay Kit (Promega) per the manufacturer’s instructions and was normalized to β-galactosidase activity.  β-galactosidase activity was determined using the o-nitrophenyl β-D-galactopyranoside (ONPG) assay (Sigma).   110 mg of ONPG was dissolved in 100 mL of 0.1 M NaH2PO4 (6.84 mL 1 M Na2HPO4, 3.16 mL 1 M NaH2PO4, 90 mL ddH2O).  Twenty microliters of cell lysate was mixed with 200 μL 0.1 M NaH2PO4 and incubated for 2-4 hours at 37°C.  After incubation, ONPG assay was read at 420 nm.  Data is presented as the mean ± standard error of the mean (S.E.M.).  Statistical significance was calculated using the Student’s T-test.  A p-value of less than 0.05 was considered significant and is denoted with an asterisk (*).

References

1 D. E. Brobst, X. Ding, K. L. Creech, B. Goodwin, B. Kelley, and J. L. Staudinger, 'Guggulsterone Activates Multiple Nuclear Receptors and Induces Cyp3a Gene Expression through the Pregnane X Receptor', J Pharmacol Exp Ther, 310 (2004), 528-35.

2 G. Hu, C. Xu, and J. L. Staudinger, 'Pregnane X Receptor Is Sumoylated to Repress the Inflammatory Response', J Pharmacol Exp Ther, 335 (2010), 342-50.

-top-


One of 34 U.S. public institutions in the prestigious Association of American Universities
Nearly $290 million in financial aid annually
44 nationally ranked graduate programs.
—U.S. News & World Report
Top 50 nationwide for size of library collection.
—ALA
23rd nationwide for service to veterans —"Best for Vets," Military Times
KU Today