Figure 1.
Sequence alignment of TLX LBD with COUP-TFII, RXRα and PNR LBDs.
The nomenclature of the helices and β-sheet is indicated. Predicted residues belonging to the LBP are indicated by pink stars. Residues involved in the binding of TLX corepressors are highlighted in yellow. An exposed cysteine C338 is highlighted in green.
Figure 2.
Models of TLX LBD and TLX LBP.
A. This panel represents TLX LBD in a putative agonist conformation. Helix α11 is in blue/green, helix α12 in orange/yellow. The LBP appears in pink circles behind helix α12. B. This panel illustrates the key amino-acids inside the LBP, hydrophobic residues are in yellow, hydrophilic residues in red.
Figure 3.
Biochemical characterization of purified TLX LBD. A.
Analytical ultracentrifugation sedimentation analysis on purified TLX LBD. Analytical ultracentrifugation sedimentation equilibrium profiles were recorded at 4°C after 70 hour incubation at three rotor speeds: 11,000 r.p.m. (cyan), 18,000 r.p.m. (green) and 22,000 r.p.m. (blue), for a 10 µM TLX sample, dissolved in 20 mM Tris-HCl, 150 mM NaCl, 5 mM DTT, 10% (v/v) glycerol, 1% (v/v) DMSO and 2 mM CHAPS at pH 8.0. The upper panel shows the sedimentation equilibrium profiles with the lines of best fit shown in red. The best fit was obtained for a monomer-dimer equilibrium model with a Kd of 10 µM. 95% confidence interval limits for this dissociation constant were determined to be 4 µM<Kd<24 µM. The lower panel provides, for each dataset, the residuals for the data fitting. This is an illustration on how accurate the fitting actually is. B. This panel represents the SDS page gel for the pooled fractions of purified TLX LBD protein after size-exclusion chromatography (first column, shown with the asterisk). The protein ladder appears in the second column. In presence of this denaturing gel, TLX LBD runs at a molecular weight of 25 KDa, corresponding to the molecular weight for the monomeric TLX LBD. C. This panel is an illustration of the size-exclusion calibration curve. TLX LBD elutes at a volume corresponding to a dimer. In the inset window is shown the size-exclusion chromatogram for TLX LBD. TLX LBD elutes as a sharp symmetric peak.
Figure 4.
Results of direct binding assays using the DSF method.
A. This panel represents the melting curve of purified TLX LBD in the presence of 5% DMSO. B. This panel allows a rapid visualization on how compounds binding to TLX LBD can shift the reference Tm. We plotted the first derivatives of the melting curves for unliganded TLX LBD and TLX LBD in presence of ccrp1, ccrp2 and ccrp3. While these curves were utilized to accurately calculate Tm, a rough approximation of the Tm would be the minimum of the first derivative curves. In presence of the three compounds this minimum shifted downward. C. This panel displays the chemical structures of ccrp1, ccrp2, and ccrp3.
Table 1.
Description of the TLX ligands.
Figure 5.
Results of direct binding assays for ccrp1, ccrp2 and ccrp3 on the TLX LBD using the Octet RED 384 instrument.
A, B, C. Those panels represent the plotted steady-state response levels and the fitted binding isotherms. The purified TLX protein was immobilized onto the surfaces of Super-Streptavidin biosensors. Solutions of compounds ccrp1 (panel A), ccrp2 (panel B), ccrp3 (panel C) at 0.4–100 µM concentrations were tested against immobilized TLX LBD and reference surfaces composed of blocked biotinylated Streptavidin.
Figure 6.
Modulation of TLX and PNR transcriptional activities by ccrp1, ccrp2 and ccrp3. A.
Transfections of TLX LBD repress the UAS promoter leading to a decrease in luciferase activities compared to the control (cells transfected with empty GAL4 vector). Compounds ccrp1, ccrp2 and ccrp3 respectively enhance repressive transcriptional activity of TLX only in cells transfected with TLX LBD. HeLa cells transiently transfected with TLX LBD or empty GAL4 vector and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at different concentrations (indicated). Following 16 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines. B. HeLa cells transiently transfected with PNR LBD or empty GAL4 vector and the luciferase reporter gene were treated with DMSO (0.1%, solvent control) or ccrp1, ccrp2 and ccrp3 at 10 µM. Following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown as fold repression relative to control (cells transfected with empty GAL4 vector and treated with 0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.
Figure 7.
Specificity of ccp1, ccrp2 and ccrp3 towards unrelated nuclear receptors.
A. This panel represents the melting curve of purified ERβ LBD in the absence of ligand. B. Melting temperature shifts for ERβ LBD in absence of compounds or treated with estradiol (E2), ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by estradiol. C. This panel represents the melting curve of purified LXRβ LBD in the absence of ligand. D. Melting temperature shifts for LXRβ LBD in absence of compounds or treated with T0901317, ccrp1, ccrp2 and ccrp3 at a final concentration of 500 µM. Neither compound demonstrates any significant effect on the melting temperatures compared to the shift induced by T0901317.
Figure 8.
Modulation of RXRα, COUP-TFII and ERβ transcriptional activities by ccrp1, ccrp2 and ccrp3.
A, B. HeLa cells transiently transfected with RXRα LBD (panel A) or COUP-TFII LBD (panel B) and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or compounds of interest at 10 µM in absence or presence of 9-cis retinoic acid (RA) at different concentrations (100 nM for RXRα and 5 µM for COUP-TFII). C. HeLa cells transiently transfected with ERβ LBD and the luciferase reporter gene were treated with either DMSO (0.1%, solvent control) or ccrp1; ccrp2 and ccrp3 at 10 µM in absence or presence of E2 at 100 nM. For all panels, following 24 h treatments, luciferase activities were recorded and normalized. For each concentration point, data are shown relative to control (0.1% DMSO), as average of three independent measurements, with experimental errors shown as black lines.