Page 76 - GPD-2-3

Gene & Protein in Disease Hotspots in the FOXO4: p53 interaction

same sequence in wild-type FOXO4. This suggests that the β-mercaptoethanol, and 500 mM imidazole). Fractions
interactions of the DRI may not be similar to those of the containing the target protein were identified using SDS-
equivalent FOXO4 sequence in the wild-type FOXO4:p53 PAGE and subsequently pooled and concentrated using an
complex. These differences in molecular features are likely Amicon Ultra-15 concentration unit. Further purification
to strongly impact the interactions between the DRI and was carried out using a Superdex 75 (GE Healthcare)
its p53 target. column with SEC buffer (20 mM Bis-Tris at pH 7.8, 150
In this study, we explored whether the differences in mM NaCl, and 10 mM β-mercaptoethanol). Fractions
binding energy could be determined between the DRI and containing the protein of interest were once again
its L-amino peptide counterpart in relation to the p53- identified using SDS-PAGE, pooled, and concentrated to
DBD. In addition, we present the use of an in silico model a final concentration of 10 mg/ml. The samples were then
for the DRI: FOXO4 interaction, which generated data flash-frozen in liquid nitrogen and stored at −80°C until
on the likely key interactions between FOXO4 and p53- further use.
DBD. This model aided in the design of shorter peptides to 2.2. Peptides synthesis and characterization
test hypotheses generated from the model. Subsequent in
vitro experiments demonstrated that the newly developed All peptides (Table 1) were synthesized using
shorter peptides contain the key interaction elements, as solid-phase peptide synthesis, following the Fmoc
they retain almost complete binding affinity of the DRI to (N-9- fluorenylmethyloxycarbonyl) strategy as described
[19,20]
p53-DBD. These experiments provide data to aid in the in the literature . The synthesis was carried out on
design of smaller molecules (cyclic peptides or synthetic Wang resin (loading at 0.3 mmol/g for long peptides and
molecules) aimed at improving the pharmacological 1.2 mmol/g for short ones) with a fully automated peptide
properties of inhibitors targeting the p53:FOXO4 PPI. synthesizer, Syro I from MultiSynTech GmbH (Witten,
Germany). Each peptide was cleaved from the solid
2. Materials and methods support using a mixture composed of 90% trifluoroacetic
acid (TFA), 5% triisopropylsilane (TIS), and 5% H2O
2.1. Protein expression and purification (v/v/v) and stirred for about 3 h at room temperature.

The p53-DBD gene was cloned into the pETM-11 The resulting peptides were precipitated in cold diethyl
[18]
expression vector using the NcoI and HindIII restriction ether, and the pellets were resuspended in a mixture of
sites and confirmed by sequencing. A single colony was water and acetonitrile (H O/ACN = 75:25 v/v) and then
2
inoculated into 10 ml LB culture medium containing 50 lyophilized. Peptide purification was carried out using a
μg/ml kanamycin and 35 μg/ml chloramphenicol. This NUCLEODUR HTec C18 column (5 μm, 150×21 mm) at
culture was incubated overnight at 37°C with shaking at a flow rate of 12 ml/min. Elution was carried out using a
180 rpm. Subsequently, the 10 ml overnight culture was gradient of solvent B (CH CN + 0.1% TFA) from 5% to
3
transferred into 1 L of LB medium supplemented with 70% over 20 min, with solvent A (H2O+0.1% TFA) as the
50 μg/ml kanamycin and 35 μg/ml chloramphenicol. The other component. The purity and identity of the peptides
culture was maintained at 37°C with continuous shaking were verified using analytical liquid chromatography with
at 180 rpm until the OD600 reached approximately 0.6. mass spectrometry (LC-MS), and the results are presented
At this point, isopropyl β-D-1-thiogalactopyranoside in Table 1. LC-MS analysis of peptides was carried out
(IPTG) was added to a final concentration of 0.5 mM, and using a C18 Waters XBridge column (3 μm, 4.6 × 5.0 mm).
the culture was further incubated at 18°C for 16 h. Cells A linear gradient of 0.05% TFA in CH CN from 5 to 70%
3
were harvested by centrifuging the culture at 6000 rpm over 15 min was applied at a flow rate of 0.2 ml/min. The
for 30 min at 4°C and then resuspended in lysis buffer purity of all peptides exceeded 95% and determined by
(100 mM HEPES at pH 7.5, 100 mM NaCl, and 5 mM calculating the ratio of the peak area of the target peptide
β-mercaptoethanol). The cells were sonicated in an ice- to the sum of all areas in UV chromatograms at 210 nm.
water bath for 6 min. The supernatant was collected through
high-speed centrifugation at 18000 rpm for 45 min at 4°C 2.3. Microscale thermophoresis
and subsequently loaded onto a 5 ml HisTrap HP (Cytiva) The binding affinity of peptides toward p53 was analyzed
column that had been pre-equilibrated with lysis buffer. using the microscale thermophoresis (MST) technique.
The column was then washed with 10 column volumes For the MST experiments, purified p53 protein was diluted
of washing buffer (100 mM HEPES at pH 7.5, 100 mM to a concentration of 50 nM and labeled using the His-
NaCl, 5 mM β-mercaptoethanol, and 25 mM imidazole). Tag Labeling Kit RED-tris-NTA (Monolith Cat#-L018),
Protein was eluted using a linear gradient with an elution following the manufacturer’s recommended instructions. The
™
buffer (100 mM HEPES at pH 7.5, 100 mM NaCl, 5mM labeled protein was pretested using Monolith NT.115 MST

Volume 2 Issue 3 (2023) 3 https://doi.org/10.36922/gpd.1491