DARPins as a novel tool to detect and degrade p73

Protein expression and purification

DARPins and p53 family domains were expressed in E. coli and purified as described before [29, 30]. In brief, E. coli BL21(DE3) Rosetta cells were transformed with the respective pET-15b expression plasmid. Cells were grown in 2xYT medium until an optical density of 0.8 was reached. Protein expression was induced with 0.6 mM IPTG for 16 h at 16 °C. Cells were harvested by centrifugation, resuspended in IMAC A buffer (50 mM Tris pH 8, 400 mM NaCl, 20 mM β-mercaptoethanol) supplemented with RNAse (Sigma), DNAse (Sigma), lysozyme (Sigma), and self-made protease inhibitors, and were lysed by sonification. After clearing the lysate by centrifugation, the supernatant was supplemented with 30 mM imidazole and loaded on a pre-equilibrated immobilized metal ion affinity chromatography (IMAC) column (HiTrap IMAC Sepharose FF, Cytiva) following an IMAC purification protocol. The eluted protein was simultaneously dialyzed to IMAC A buffer and digested with TEV protease (home-made). TEV protease and undigested protein were separated by a reverse IMAC step. The purified proteins were further purified and buffer exchanged by size-exclusion chromatography (SEC) with SEC buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.5 mM TCEP) using a Superdex 75 10/300 column (Cytiva) using an ÄKTA purifier system at 4 °C. Central peak fractions were collected, concentrated to a concentration 300 to 500 µM (Amicon Ultra Centrifugal Filters, Millipore) and flash-frozen in liquid nitrogen prior to storage at -80 °C until use. The purity and molecular weight of purified proteins were monitored by SDS-PAGE and LC-ESI-TOF-mass spectrometry.

DARPin biotinylation

Avi-tagged target proteins for DARPin selections and DARPins containing an N-terminal Avi-tag were enzymatically biotinylated in vitro using the E. coli biotin ligase BirA. BirA was subcloned into a pET-15b-GFP-His8-TEV E. coli expression vector (Novagen, Merck KGaA) and was expressed and purified as described before [29]. The in vitro biotinylation was performed by mixing GFP-BirA in a 1:50 molar ratio with the respective Avi-tagged DARPin in SEC buffer supplemented with 10 mM ATP, 10 mM MgCl₂, 0.5 mM biotin. The mixture was incubated for 16 h at 16 °C and subsequently separated SEC using a Superdex 75 10/300 column on an ÄKTA Purifier system (GE Healthcare). Biotinylated DARPin fractions were pooled and analyzed by LC-ESI-TOF-mass spectrometry. Only DARPins showing close to 100% labeling efficacy were used for experiments.

Selection and screening of DARPin binders

To generate DARPin binders specific for p73, either biotinylated p73 OD (aa 351–398 of TAp73α) or the p73 SAM domain (aa 489–550 of TAp73α) was immobilized on either MyOne T1 streptavidin-coated beads (SA; Pierce) or Sera-Mag neutravidin-coated beads (NA, GE), depending on the particular selection round, and these beads were alternated. Ribosome display selections were performed essentially as described [19, 20], but using a semi-automatic KingFisher Flex MTP96 well platform.

The library includes N3C-DARPins with the original randomization strategy as reported [31] but uses a stabilized C-cap [16, 45, 46]. Additionally, the library is a mixture of DARPins with randomized and non-randomized N- and C- terminal caps, respectively [16, 47]. Successively enriched pools were cloned as intermediates in a ribosome display-specific vector [47]. Selections were performed over four rounds with decreasing target concentration and increasing washing steps to enrich for binders with high affinities. In addition, a prepanning with BSA blocked SA or NA beads was performed to eliminate unspecific DARPins in rounds two to four in the selection for p73 OD.

The final enriched pool was cloned as fusion construct into a bacterial pQE30 derivative vector with a N-terminal MRGS(H)8 tag and C-terminal FLAG tag via unique BamHI x HindIII sites containing a T5lac promoter and lacIq for expression control. After the transformation of E. coli XL1-blue, 380 single DARPin clones were expressed and tested for target binding in a Homogeneous Time Resolved Fluorescence (HTRF)- or ELISA-based assay From the identified binders, 32 random target-specific clones for both targets were sequenced and single clones identified. For the selection of DARPins against the p73 SAM domain (aa 489–550) DARPins, 21 DARPins were single clones and unique. Out of this panel, clone B9 described in this study was derived. For the selection of DARPins against the p73 OD DARPins, clone 1800 described in this study was obtained only after monoclonalization of a double clone.

The final enriched pool was cloned as fusion construct into a bacterial pQE30 derivative vector with a N-terminal MRGS(H)8 tag and C-terminal FLAG tag via unique BamHI x HindIII sites containing a T5lac promoter and lacIq for expression control.

After the transformation of E. coli XL1-blue, 380 single DARPin clones for p73 OD or the p73 SAM domain (aa 489–550) were expressed in 1‑mL scale in deep-well plates by addition of IPTG (isopropyl β-D-1-thiogalactopyranoside). Cells were harvested by centrifugation, and lysed with the addition of B-Per Direct detergent plus Lysozyme and Nuclease (Pierce). The lysates were cleared by centrifugation. These bacterial crude extracts of single DARPin clones were subsequently used in a Homogeneous Time Resolved Fluorescence (HTRF)-based screen for target p73 OD, while for target p73 SAM domain (aa 489–550) an ELISA-based assay was used to identify potential binders. In HTRF, binding of the FLAG-tagged DARPins to streptavidin-immobilized biotinylated p73 OD was measured using FRET (donor: Streptavidin-Tb cryptate (610SATLB, Cisbio), acceptor: mAb anti-FLAG M2-d2 (61FG2DLB, Cisbio). Further HTRF measurement against ‘No Target’ allowed for discrimination of p73 (aa 112–594))-specific hits. Experiments were performed at room temperature in white 384-well Optiplate plates (PerkinElmer) using the Taglite assay buffer (Cisbio) at a final volume of 20 μl per well. FRET signals were recorded after an incubation time of 30 min using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm) and multiplying this value by 10,000 to derive the 665/620 ratio. The background signal was determined by using reagents in the absence of DARPins. In ELISA, binding of the FLAG-tagged DARPins to neutravidin-immobilized biotinylated p73 SAM domain (aa 489–550) was detected using a mouse monoclonal anti-FLAG-M2 antibody (Sigma, F3165) and a goat-anti-mouse antibody coupled to alkaline phosphatase as secondary antibody (Sigma, A3562). Target-specific binding of DARPins was analyzed by following the hydrolysis of para-nitrophenylphosphate at 405 nm in an ELISA-plate reader (Tecan). The background signal was determined by using reagents in the absence of biotinylated target protein.

From the identified binders, 32 random target-specific clones for both targets were sequenced and single clones identified. For the selection of DARPins against the p73 SAM domain (aa 489–550) DARPins, 21 DARPins were single clones and unique. These DARPins were characterized regarding their specificity towards p73 by pulldown assays with isoforms of all p53 family members yielding DARPin 1800 as a specific binder for the p73 OD and DARPin B9 as a specific binder for the p73 SAM domain.

Molecular cloning

For recombinant protein expression of DARPins and all p63/p73 constructs, derivatives of a pET15b plasmid (Novagen, Merck KGaA) were used. Inserts were generated by PCR and introduced into pET15b-His₁₀-TEV, pET15b-His₁₀-TEV-Avi or pET15b-His₁₀-TEV-HA (N-terminal His₁₀-tag followed by a tobacco etch virus (TEV) protease cleavage site (and Avi- or HA-tag)) by subcloning using BamHI and XhoI restriction sites. For transient expression in mammalian cell culture, PCR-generated inserts were introduced in the pcDNA3.1(+) Myc plasmid (Invitrogen, Thermo Fisher Scientific Inc) by subcloning using BamHI and XhoI resctriction sites. For the generation of stable cell lines, PCR-generated inserts of p63 or p73 isoforms were introduced in a pcDNA5 FRT/TO plasmid by subcloning using BamHI and XhoI resctriction sites.

For generation of the leucine zipper DARPin constructs the protein coding sequence including the DARPins C-terminally fused to a (G₄S)₄-linker followed by the leucine zipper sequence (amino acids 250–281 of yeast GCN4) was synthesized (Eurofins). This gene strand was inserted into a pET15b-His₁₀-TEV, pET15b-His₁₀-TEV-Avi or pET15b-His₁₀-TEV-HA by subcloning using BamHI and XbaI resctriction sites.

For generation of DARPin-E3 chimeras, a PCR generated insert of the respective E3 ligase was introduced into the pcDNA3.1 (+) HA plasmid (Invitrogen, Thermo Fisher Scientific Inc) using XhoI and XbaI. Subsequently, the respective DARPins were introduced by subcloning with BamHI and XhoI.

Cell culture

The non-small cell lung cancer cell line H1299 (ATCC, CRL5803) was cultured in RPMI medium 1640 (Gibco), containing 10% FBS (Capricorn Scientific), 100 U/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco) at 37 °C and 5% CO₂. For recombinant protein expression, the H1299 culturing medium was exchanged with antibiotic-free medium, and cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Six hours after transfection the medium was exchanged to standard H1299 culturing medium.

For a generation of stable cell lines inducible expressing TAp73α, ∆Np73α or ∆Np63α, the Flp-In T-REx system (Thermo Fisher Scientific) was used. The T-REx-U-2 OS cell line was cultured for two weeks in DMEM medium (Gibco), containing 10% FBS (Capricorn Scientific), 4 μg/ml blasticidin (Gibco), 333 μg/ml Zeocin (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 1 mM pyruvate (Gibco) at 37 °C and 5% CO₂. T-REx-U-2 OS cells were transferred into six-well plates for transfection using Lipofectamin 2000 transfection reagent (Thermo Fisher scientific) and were transfected with pcDNA5/FRT/TO plasmid (Thermo Fisher Scientific) containing either TAp73α, ∆Np73α or ∆Np63α, and the pOG44 plasmid (Thermo Fisher Scientific) expressing the Flp recombinase according to the manufacturer’s instructions. After transfection, cells were cultured in DMEM containing 10% tetracycline-free FBS (Bio Cell) for 48 h and re-seeded into 15 cm dishes. Twenty-four hours after re-seeding, the medium was exchanged to selection medium (DMEM containing 10% tetracycline-free FBS, 4 μg/ml blasticidin, 200 μg/ml hygromycin (Thermo Fisher Scientific), 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mM pyruvate) and cells were cultured until a non-transfected control showed no viable cells (about 10–14 days). For monoclone selection, twelve single colonies of each cell line were isolated and expression of target proteins was induced by the addition of 1 µg/mL tetracycline (Thermo Fisher Scientific) to the selection medium. After twenty-four hours, the expression of target proteins was analyzed by Western Blot. The best three monoclones were selected for further experiments.

The H1299 cell line was obtained from ATCC. The T-REx-U-2 OS cell line was a gift from Christian Behrends (Ludwig-Maximilians-University (LMU), Munich, Germany). All cell lines used in this study were routinely tested for mycoplasma contaminations.

Gel electrophoresis and western blotting

Purified proteins were combined with SDS loading buffer (containing 250 mM Tris, pH 8.0, 7.5% (w/v) SDS, 25% (w/v) glycerol, 12.5% (v/v) β-mercaptoethanol, and 0.025% (w/v) bromophenol blue), subjected to denaturation at 95 °C, and then separated on manually prepared 4–16% Tris-glycine gels. The gels were subsequently stained using Quick Coomassie Stain (NeoBiotech) following the manufacturer’s guidelines.

For immunoblotting, samples were either mixed with SDS loading buffer or NuPAGE LDS buffer (Thermo Fisher Scientific) supplemented with DTT, denatured at 95 °C, and applied onto 4–15% Mini-PROTEAN TGX Stain-Free Precast Protein gels (Bio-Rad). Gel transfer was carried out using the TransBlot Turbo Transfer System (Bio-Rad) as per the manufacturer’s instructions. Membranes were blocked for 1 h in a blocking buffer (TBS, 0.05% (v/v) Tween-20, 5% skim milk powder, Sigma-Aldrich) and then incubated with the primary antibody in a blocking buffer overnight with shaking at 4°C. Following three washes with TBS-T, membranes were incubated with the secondary antibody in a blocking buffer under shaking conditions for 1 h at room temperature. Subsequently, membranes underwent three additional washes with TBS-T and were analyzed by adding Amersham ECL Prime WB Detection Reagent (Cytiva). Western blot signal quantification was performed using ImageLab (version 6.1, Bio-Rad).

The following antibodies and dilutions were used: anti-myc (1:2000, clone 4A6, Millipore), anti-p73 (1:2000, ab40658, Abcam), anti-HA (1:1000, clone 16B12, BioLegend) anti-vinculin (1:2000, clone 7F9, Santa Cruz Biotechnology), goat anti-mouse HRP (1:5000, A9917, Sigma Aldrich), goat anti-rabbit HRP (1:5000, 111-035-144, Jackson ImmunoResearch).

Pulldown assays

Recombinant target proteins (cloned into a pcDNA3 vector) were expressed in H1299 cells. Endogenous target proteins were pulled down from tissues samples of eight-day-old (P8) female CD-1 mice, purchased from Charles River Laboratories. Cell and tissue lysates for pulldown samples were generated as described before [29]. Magnetic Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific) were pre-incubated with an excess of biotinylated DARPin in Pulldown (PD) wash buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.1% (v/v) Tween-20) while rotating for 1 h at 4 °C. Subsequently, the beads were washed three times with PD wash buffer and were resuspended in the same volume of PD wash buffer as before to maintain magnetic beads concentrations. For PD experiments, 10 µl DARPin loaded beads were mixed with freshly prepared cell lysate or tissue lysate and the total volume was adjusted to 1000 µl with pD wash buffer supplemented with 1x Complete Protease inhibitor (Roche). The PD samples were incubated rotating overnight at 4 °C. The beads were washed five times with 1000 µl PD wash buffer and bound proteins were eluted by boiling in 50 µl LDS buffer at 70 °C for 10 min. Samples were analyzed by Western Blot. Myc-tagged recombinant target proteins were detected using a mouse anti-myc antibody (1:2000, clone 4A6, Millipore). Endogenous p73 was detected using a rabbit anti-p73 antibody (1:1000, ab40658, Abcam).

Isothermal titration calorimetry

All titration experiments were performed using a MicroCal VP-ITC microcalorimeter (Malvern Instruments Ltd, UK). DARPins and target proteins were dialyzed against ITC buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM TCEP). Target proteins were titrated to constant concentrations of DARPin in 10 μl steps with total injections of 25 and 250 s spacing time at indicated temperatures. The reference power was set to 25 μCal/s and stirring speed to 307 rpm. Automated unbiased baseline calculation and curve integration was done with NITPIC [48]. Thermodynamic parameters and the equilibrium constant were calculated by SEDPHAT [49]. The first data point was always excluded from the analysis.

Protein crystallization and structure determination

Protein complexes consisting of DARPin and the respective p73 domain were prepared by mixing the purified proteins in SEC buffer. In case of the p73 SAM complex, proteins were mixed in 1:1 molar ratio. For the p73 OD complex, a 1:2 molar ratio (OD:DARPin) was used. After overnight incubation at 16 °C, unbound proteins were separated from the complex by SEC in crystallization buffer (20 mM Tris pH 7.8, 50 mM NaCl, 0.5 mM TCEP) using a Superdex 75 10/300 column. The central peak fractions corresponding to the complex were pooled and concentrated to a final complex concentration of ~300 µM. Protein purity was assessed by SDS-PAGE and LC-ESI-TOF-MS. Crystals were grown at 20 °C using the sitting drop vapor diffusion and the condition containing 30% PEG2000MME and 0.15 M potassium bromide. Crystals were cryo-protected with mother liquor containing 22% glycerol prior to flash-freezing in liquid nitrogen. Diffraction data were collected at the Swiss light source (Villigen, Switzerland). The obtained diffraction data were integraded with the program XDS⁴² and scaled with AIMLESS⁴³. The crystal structures of the complexes were solved by molecular replacement using Phaser⁴⁴ with published structures of the p73 OD (PDB: 5HOB) and DARPin (PDB: 6FP7) as search models. In the case of the p73 SAM domain-DARPin B9 structure a single DARPin (PDB: 6FP7) was used as a search model. Model building and refinement was performed using COOT ⁴⁵ and REFMAC5⁴⁶. X-ray data collection and refinement statistics are listed in Supplementary Table S3. Structural figures in this paper were prepared using PyMol (pymol.org).

Immunofluorescence staining

Stable T-REx-U-2 OS cell lines expressing TAp73α, ∆Np73α or ∆Np63α were seeded on cover slips (Carl Roth). Expression of target genes was induced by addition of 1 µg/mL tetracycline (Thermo Fisher Scientific) to the culture medium. Twenty-four hours after induction of protein expression, cells were washed twice with PBS and fixed with Roti Histofix 4% (Carl Roth) for 10 min at room temperature. Fixed cells were washed twice with PBS, followed by permeabilization with PBS-T (PBS supplemented with 0.1% Triton X-100, Carl-Roth) for two times 5 min at room temperature. Permeabilized cells were blocked in blocking buffer (PBS-T supplemented with 1% BSA, Carl Roth) for 20 min at room temperature. Afterwards, cells were incubated with 100 nM HA-tagged DARPin or HA-tagged Leucine Zipper DARPin and mouse anti-myc antibody (1:500, clone 4A6, Millipore) in blocking buffer overnight at 4 °C. Cells were washed five times with PBS-T and incubated with Alexa Fluor 568 anti-goat antibody (1:200, A11057, Life Technologies) and Alexa Fluor 647 anti-mouse antibody (1:200, A31571, Life Technologies) in blocking buffer for 2 h at room temperature. Slides were washed five times with PBS-T and coverslips were mounted using Mowiol (Carl Roth) mounting medium which was supplemented with DAPI (Thermo Fisher Scientific). A detailed recipe of the mounting medium can be found at CSH protocols (http://cshprotocols.cshlp.org/content/2006/1/pdb. rec10255). The slides were dried several days before imaging with a LSM 780 confocal laser scanning microscope (Zeiss).

Protein degradation assay

To investigate the degradation of target proteins by DARPin-E3 chimeras, H1299 cells were seeded into white Nunc 96-well microplates (Thermo Fisher Scientific) in triplicates. Twenty-four hours after seeding, the cells were transfected with plasmids containing the DARPin-E3 chimera were co-transfected with the pBit4.1-N[HiBit-IRES-luc2/CMV/Blast] vector containing the respective p63 or p73 isoform using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The firefly and Nanoluc luciferase activity was measured on a Spark plate reader (Tecan) using the Nano-Glo HiBit Dual-Luciferase Reporter System (Promega) according to the manufacturer’s recommendations. The experiment was repeated in three biological replicates. The protein level was determined by calculating the ratio of Nanoluc to firefly luciferase activity which was normalized to a sample not containing any DARPin-E3 chimera. Statistical significance was analyzed by ordinary one-way ANOVA (n.s.: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001) using Prism (Version 8.2.1, GraphPad).

Proteasome inhibition assay

Stable U-2 OS cells expressing TAp73α were seeded into 12-well plates. Expression was induced by supplementation of the culture medium with 1 µg/mL tetracycline (Thermo Fisher Scientific). Eight hours after induction of protein expression medium was exchanged to culture medium without antibiotics but supplemented with tetracycline, and cells were transfected with pcDNA3.1 (+) HA-NLS plasmids with the respective DARPin-E3 chimera N-terminally tagged with an HA-tag followed by a nuclear localization signal (NLS, sequence: KKKRKV). A sample transfected with empty pcDNA3.1 (+) served as negative control. Sixteen hours after transfection cells were treated with DMSO (negative control) or the proteasome inhibitor bortezomib (100 nM) for 6 h. Cells were detached with Accutase and resuspended in 1x LDS sample buffer followed by Western Blot analysis using the anti-myc (1:2000, clone 4A6, Millipore) antibody.

Transactivation assay

For transactivation assays, H1299 cells were seeded in 12-well plates and transfected using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. All transfection mixes contained 200 ng of pRL-CMV plasmid (Promega) for constitutive expression of Renilla luciferase, 200 ng of a reporter plasmid with firefly luciferase under the control of the artificial pBDS-promotor (3 copies of the wt p53 binding site from the 14-3-3σ promotor) and varying amounts of pcDNA3.1 (+) HA-NLS with the respective DARPin-E3 chimera N-terminally tagged. The pBV-Luc BDS-2 3x WT plasmid (addgene plasmid #16515) was a gift from Bert Vogelstein [50]. In the case of the promotor squelching assay, the transfection mix contained 50 ng Myc-tagged ∆Np73α and 10 ng Myc-tagged p53 as well as the reporter plasmids and the DARPin-E3 chimera plasmid. For each assay, an empty vector control only comprising empty pcDNA3.1(+) was transfected to determine the fold induction without the presence of any DARPin. Twenty-four after transfection, cells were washed with PBS (Gibco), detached with Accutase and re-seeded into white Nunc 96-well microplates (Thermo Fisher Scientific) in quadruplicates. The assay was performed using the Dual-Glo Luciferase reporter assay kit (Promega) according to the manufacturer’s instructions, and firefly as well as Renilla luciferase fluorescence was measured using a Spark plate reader (Tecan). The remaining sample was centrifuged for 5 min at 500 g, pelleted cells were mixed with 1x SDS-loading buffer, and p53 protein levels were analyzed by western blot using the anti-myc (1:2000, clone 4A6, Millipore) antibody. The experiment was repeated in three biological replicates, and the ratio of firefly to Renilla luciferase signal was normalized to empty vector control for each biological replicate. Statistical significance was assessed by ordinary one-way ANOVA (n.s.: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001) using Prism (Version 8.2.1, GraphPad).

Statistics and reproducibility

Pulldown experiments, degradation assays and transactivation assays were performed in biological triplicates. All data points are shown in the corresponding figures with the bar diagrams presenting the mean value and the error bar presenting the SD. ITC measurements were performed twice, but the determination of the K_D value is based on a single measurement. The K_D values and the 95% confidence interval were determined by SEDPHAT [49]. Immunofluorescence stainings were performed in biological duplicates with exemplary images of one replicate shown in the respective figure.

Ethical statement

All methods were performed in accordance with the relevant guidelines and Regulations. As this is an in vitro study without human patient material or experiments with animals no ethics committee approval was required.

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• Source: https://www.nature.com/articles/s41419-024-07304-2