edoc

Regulation of scaffold protein PDZK1 and its impact on the drug transporter OATP2B1

Ferreira, Célio José. Regulation of scaffold protein PDZK1 and its impact on the drug transporter OATP2B1. 2018, Doctoral Thesis, University of Basel, Faculty of Science.

[img] PDF
Restricted to Repository staff only until 5 May 2020.

48Mb

Official URL: http://edoc.unibas.ch/diss/DissB_12668

Downloads: Statistics Overview

Abstract

The PDZ domain containing 1 (PDZK1) is a scaffold and PDZ protein of the Na+/H +exchanger regulatory
factor (NHERF) family. PDZ proteins consist of one or more PDZ domains that recognizes specific amino acid
sequences at the C-terminus of membrane proteins such as transporters or receptors. PDZK1 harbors four PDZ
domains that organize multiprotein complexes by protein-protein interaction whereby stabilizing the targeted proteins
at the membrane and modulating their function due to the spatial organization of the interaction partners. Transporters
influenced by PDZK1 are members of the solute carrier (SLC) or the ATP binding cassette (ABC) superfamily, which
are associated to drug metabolism and to the cellular uptake or efflux of endogenous substrates of the thyroid and uric
acid homoeostasis.
Genome wide association studies (GWAS) reported an association between single nucleotide polymorphisms
(SNP) located in the promoter of scaffold protein and uric acid levels in the tested patients. Interestingly, many of the
transporters involved in the handling of the uric acid such as SLC22A12 (URAT1) or ABCC4 (MRP4) were previously
shown to directly interact with PDZK1. These data created the hypothesis of a “urate transportosome”, a network with
PDZK1 orchestrating a multi protein complex of influx and efflux transporters responsible for handling uric acid in
the proximal tubulus of the kidney. A central transcriptional factor of the proteins in a “transportosome” maybe a
possibility to synchronize a functional unit like the “urate transportosome” to achieve a coordinated secretion and
absorption of urate.
In chapter 3.1 we generated further evidence for the “urate transportosome” by reporting a correlation
between the expression of urate transporters and of PDZK1 and of the hepatocyte nuclear factor 1 a (HNF1a), a
transcription factor. Additionally, HNF1a was shown to regulate PDZK1 expression. First, we observed a
transactivation of the promoter of the scaffold protein in presence of HNF1a. Furthermore, the overexpression of the
homoebox in primary kidney cell (RPTEC) augmented the mRNA and the protein content of PDZK1. Deletion of the
most likely HNF1a binding site on the PDZK1 promoter as well as the ChIP assay verified the direct interaction of
the transcription factor with the scaffold protein,
The presence of PDZK1 is not limited to the apical membrane of the proximal tubulus cells in the kidney
although it is the tissue with the highest expression. The scaffold protein was also detected in hepatocytes. One of the
major focus concerning PDZK1 and its expression at the sinusoidal membrane of the hepatocytes is the involvement
in the reverse cholesterol transport by its influence on the Scavenger Receptor B 1 (SR-B1). SR-B1, a High-Density
Lipid protein (HDL) receptor, mediates the uptake of cholesterol from HDL particles in liver and other tissues.
Nevertheless, the treatment of mice with the TO 901317 an agonist of the central regulator of lipid homoeostasis the
liver x receptors (LXRa, NR1H3 and LXRb, NR1H2), did not affect PDZK1, but reduced the expression of SR-B1.
However, TO 901317 does not only bind to LXR but also to the nuclear receptor pregnane x receptor (PXR, NR1I2)
well known for its impact in drug metabolism.
In chapter 3.2 we investigated the role of the LXR and PXR in the regulation of PDZK1. LXR increased
while PXR reduced the promoter activity of the scaffold protein. Further, the treatment of HepG2 with a specific LXR
agonist (GW3965) increased the mRNA expression. In contrast, the exposition to TO 901317 (LXR and PXR agonist)
reduced the protein amount of PDZK1. The data of this chapter presented two new regulators of scaffold protein, LXR
a central regulator of the lipid homoeostasis and PXR involved in the protection of the organism against potential
harmful xenobiotics.
On the basis of the stabilizing effect of PDZK1 on transporters therefore increasing cellular exchange of
compounds one could assume the involvement of modulators of the metabolic rate in the transcriptional regulation of
the scaffold protein. In chapter 3.3 the thyroid hormone receptors (THRa, NR1A1 and THRb, NR1A2), which are
essential regulators of the basal metabolic rate, were reported to increase the mRNA and protein expression of PDZK1
after activation by triiodothyronine (T3) in Caco-2 cells. The binding site of THRb included one of the
abovementioned urate homoeostasis associated SNPs namely rs1967017 and the polymorphism impacted the
transactivation of the scaffold protein promoter. Additional evidence for the THRb binding site being located between
-4022bp and -4010bp of PDZK1 promoter was obtained by deletion of the binding site and a ChIP-assay.
The change of PDZK1 expression by central regulators of a gene network may indicate that other proteins
connected to this system are targeted by the scaffold protein to modulate their function. In this context regarding the
regulation of PDZK1 by thyroid hormones, the existence of PDZ binding motif at the C-terminus of the organic anion
transporting polypeptide 2B1 (OATP2B1), and the observation by Leuthold et al. that thyroxine (T4) is a substrate of
this membrane transporter, we investigated the potential modulation of OATP2B1 by the scaffold protein. The data
were summarized in chapter 3.4 showing a higher quantity of OATP2B1 at the membrane in presence of PDZK1. The
enhanced abundance of the transporter translated in a higher transporter rate (Vmax) of the canonical substrate (E1S) of
the membrane transporter. In addition, a direct interaction between both proteins was shown using the FRET method.
The importance of the PDZ binding motif of OATP2B1 for the interaction with PDZK1 was investigated by
comparison of Vmax between the wild type and OATP2B1 lacking the binding motif. The absence of PDZ binding
motif abolished the effect of PDZK1 on OATP2B1.
In chapter 3.5 we focused on the link between OATP2B1 and thyroid hormones. Previously reported data
already observed T4 as substrate of OATP2B1 and our data not only supported this finding, but also added T3 to the
compounds transported by the membrane transporter. Concerning the regulation of OATP2B1 by thyroid hormones
an increase in mRNA as well as protein expression was observed after treating Caco-2 with T3 and T4. Noteworthy
is that the regulation is likely to be cell/tissue specific since in Huh7 the presence of T3 and T4 did not affect the
mRNA and protein expression of OATP2B1.
Advisors:Meyer zu Schwabedissen, H. and Odermatt, Alex
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Biopharmazie (Meyer zu Schwabedissen)
UniBasel Contributors:Meyer zu Schwabedissen, H. and Odermatt, Alex
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12668
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (115 Seiten)
Language:English
Identification Number:
Last Modified:13 Jul 2018 04:30
Deposited On:09 Jul 2018 13:16

Repository Staff Only: item control page