p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners
- Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Germany
- Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Germany
- IMPRS on Cellular Biophysics, Germany
- Max Planck Institute for Brain Research, Germany
- Max Planck Institute of Biophysics, Germany
- Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Germany
- Fraunhofer Institute of Translational Medicine and Pharmacology, Germany
Figures
Figure 1
Domain organization of p53 isoforms.
Overview of the so far described p53 isoforms that are formed by a combination of four N-terminal (TAp53, Δ40p53, Δ133p53, and Δ160p53) and three C-terminal variants (α, β, and γ), leading to 12 individual p53 isoforms. The remaining two isoforms p53ψ (or TAp53ψ) and Δp53 (or TAΔp53α) are generated by alternative splicing.
Figure 2 with 2 supplements
Activity, oligomerization, and DNA binding.
Luciferase reporter assay of p53 isoforms and variants either on the pBDS-2 reporter (three repeats of the 14-3-3σ promoter RE) (A), or on the Mdm2 promoter (B), or on the p21 promoter (C). TAp53α carrying the cancer-related R175H mutation served as a negative control. H1299 cells (A, B) or Saos-2 cells (C) were transiently transfected with the respective luciferase reporter plasmids and the N-terminally Myc-tagged proteins. The plasmid encoding for TAp53α was titrated with the remaining DNA amount for transfection being filled up with empty vector. (D) Luciferase reporter assay of p53ψ on the pBDS-2 reporter. TAp53α carrying the cancer-related R175H mutation served as a negative control. H1299 cells were transiently transfected with the respective luciferase reporter plasmids and the N-terminally Myc-tagged proteins. (E) DNA pulldown assay of p53 isoforms with the 20 bp REs of the human PUMA and p21 promoter as bait. TAp53α carrying the cancer-related R175H mutation served as a negative control. N-terminally Myc-tagged proteins were in vitro translated using rabbit reticulocyte lysate (RRL). For the relative pulldown efficiency, each pulldown signal was normalized to the input signal. (F) Surface plasmon resonance (SPR) affinity curves of purified Δ40p53β (red), p53 DBD-OD variants (purple and black), and the isolated p53 DBD (orange) binding to the 20bp p21 response element (RE) immobilized on a streptavidin (SA) chip. Data points were extracted by equilibrium analysis of sensograms as in Figure 2—figure supplement 2, plotted and fitted with a non-linear, least squares regression using a single-exponential one-site binding model with Hill slope. p53 DBD-OD (41356) encompasses the complete OD, while p53 DBD-OD (41–331) only has the N-terminal part of the OD common to all p53α, p53β, and p53γ isoforms. (A–E) The bar diagram shows the mean values and error bars the corresponding SD (n = 3). 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).
Figure 2—figure supplement 1
Activity, of p53 isoforms.
(A) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 2A were determined by WB using an α-Myc antibody. Vinculin served as loading control. (B) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 2B were determined by WB using an α-Myc antibody. Vinculin served as loading control. (C) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 2C were determined by WB using an α-Myc antibody. Vinculin served as loading control. (D) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 2D were determined by WB using an α-Myc antibody. Vinculin served as loading control. (E) Luciferase reporter assay of p53 isoforms and variants on the p21 reporter. The same experiment as shown in Figure 2C but performed in SAOS cells instead of H1299 cells.
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Figure 2—figure supplement 1—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
DNA binding of p53 isoforms.
(A) Representative WB of the DNA pull-down assay from Figure 2E. (B) Analytical SEC of purified Δ40p53β (red), p53 DBD-OD variants (purple and black) and the isolated p53 DBD (orange). Proteins were loaded onto a Superdex 200 SEC column (24 ml bed volume) and detected by absorption at 280 nm. (C) SPR sensograms of purified Δ40p53β, p53 DBD-OD variants and the isolated p53 DBD binding to the 20bp p21 RE immobilized on a streptavidin (SA) chip. The curves were corrected for background and unspecific binding using a random DNA sequence in the reference flow cell. (D) Fit parameters from the curves in (H) are shown in the table including the SDs. Additionally, the 95% confidence interval (CI) of the KD is stated. Hill slope factor h is a measurement of cooperativity. (G-I) p53 DBD-OD (41-356) encompasses the complete OD, while p53 DBD-OD (41-331) only has the N-terminal part of the OD common to all p53α, p53β and p53γ isoforms.
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Figure 2—figure supplement 2—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig2-figsupp2-data1-v1.zip
Figure 3 with 2 supplements
Probing the DNA-binding domain (DBD) fold.
(A) Superposition of structures of the p53 DBD (grey) in complex with DNA (orange; PDB: 3TS8), HPV16 E6 (blue; PDB: 4XR8), and DARPin G4 (green; PDB: 7Z7E). Epitope of the pAB240 is marked in red and the zinc ion is depicted as a purple sphere. (B) E6 degradation assay of p53 isoforms, cancer-related mutants, and variants. ΔNp63α served as a negative control. N-terminally Myc-tagged proteins were in vitro translated using rabbit reticulocyte lysate (RRL). Lysates were diluted in reaction buffer and supplemented with either 5 µM GST-tagged HPV16 E6 or GST only as control. Reactions were incubated for 4 hr at 25°C and analysed for protein levels by WB. Signals were normalized to the loading control vinculin and the relative protein level ratio between E6 and GST samples was calculated by setting the normalized signal of the GST samples to 1. (C) Conformation-specific immunoprecipitation (Conf-IP) of p53 isoforms and cancer-related mutants. H1299 cells were transiently transfected with empty vector or N-terminally Myc-tagged p53 variants. p53 was immunoprecipitated (IP) with either α-mouse IgG or an α-p53 antibody (pAB240). The latter binds an epitope in the DBD of p53, which is only exposed when the domain is unfolded. Consequently, pAB240 only recognizes intrinsically unfolded p53 mutants under native IP conditions. Input and IP samples were subsequently analysed by WB using αMyc antibody. For the relative Conf-IP efficiency, each IP signal was normalized to the input signal. (D) DARPin pulldown assay of p53 isoforms and cancer-related mutants in vitro translated using RRL or transiently expressed in H1299 (E) with immobilized DARPin G4. DARPin G4 only recognizes the folded DBD of p53. For the relative pulldown efficiency, each pulldown signal was normalized to the input signal. Note that the expression temperature in the RRL experiment was 30°C lower than the temperature in the cell culture experiment (37°C), resulting in a higher level of folded R175H p53 mutant and hence a higher pulldown efficiency. (B–E) The bar diagram shows the mean values and error bars the corresponding SD (n = 3). 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).
Figure 3—figure supplement 1
Probing the DBD Fold by degradation via interaction with E6 protein.
(A) Representative WB of the E6 degradation assay from Figure 3B. In-vitro translated proteins were detected using an α-Myc antibody and the presence of GST and GST-E6 in the reaction mixtures was verified using an α-GST antibody. Vinculin served as a loading control.
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Figure 3—figure supplement 1—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
Co-IP experiments with different binding partners.
(A) Representative WB of the Conf-IP from Figure 3C. In-cellulo expressed proteins were detected using an α-Myc antibody. Vinculin served as loading control. (B) Representative WB of the DARPin PD in RRL from Figure 3D. In-vitro translated proteins were detected using an α-Myc antibody and the presence of DARPin G4 in the reactions was verified using an α-Strep antibody. (C) Representative WB of the DARPin PD in H1299 cells from Figure 3E. In-vitro translated proteins were detected using an α-Myc antibody and the presence of DARPin G4 in the reactions was verified using an α-Strep antibody. Vinculin served as a loading control.
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Figure 3—figure supplement 2—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig3-figsupp2-data1-v1.zip
Figure 4 with 3 supplements
Aggregation and solubility.
(A) Predicted aggregation propensity of TAp53α (black) and the C-terminal isoforms TAp53β (auburn), TAp53γ (purple), and TAp53ψ (pink). TAp53α is predicted to have a total of four aggregation-prone regions (APR1–4) sharing the first three with TAp53β and TAp53γ. TAp53γ and TAp53ψ each introduced a novel APR (APR5 and APR6) with their specific C-termini. A schematic illustration of the boundaries of all N- and C-terminal p53 isoforms is shown below the graph. Position and length of the novel sequences introduced by the C-terminal are shown with the corresponding colours. All in silico predictions were run using the TANGO algorithm with the following parameters: pH 7.5, 150 mM ionic strength and at a temperature of 37°C. (B) Sequence alignment of p53α with the p53β- and p53γ-specific C-termini. The boundaries of the OD are indicated by the grey line and the predicted APRs from (A) are highlighted in the respective colours. (C) Sequence alignment of p53α and the p53ψ-specific C-terminus. The boundaries of the DNA-binding domain (DBD) are indicated by the grey line and the predicted APRs from (A) are highlighted in the respective colours. (D) Analytical SEC of p53 isoforms and cancer-related mutants. H1299 cells were transiently transfected with the N-terminally Myc-tagged proteins and cell lysates were loaded onto a Superose 6 SEC column. Both lysis and running buffer contained 20 mM CHAPS. Collected fractions were analysed for p53 by WB using an α-Myc antibody. An elution volume of 0.820 ml corresponds to the void volume of the column (2.4 ml bed volume). (E) BN–PAGE of p53 isoforms and the cancer-related R175H mutant. H1299 cells were transiently transfected with the N-terminally Myc-tagged proteins. Cell lysates were subsequently analysed by BN–PAGE and SDS–PAGE (shown in Figure 4—figure supplement 1) followed by WB using α-Myc antibody for detection. High molecular weight species corresponding to aggregates are marked by ‘a’. (F) Solubility assay of p53 isoforms and cancer-related mutants. H1299 cells were transiently transfected with the indicated Myc-tagged proteins and lysed in a buffer supplemented with Triton X-100. Soluble and insoluble components were separated by centrifugation. The insoluble fraction in the pellet was solubilized with a buffer supplemented with SDS. Samples of both fractions were analysed by WB using αMyc antibody. (G) Thioflavin T (ThT) fluorescence assay of the p53β (aa 322–341) and p53γ (322–346) C-termini. The peptides, solubilized in denaturation buffer, and denaturation buffer only as a control, were diluted 20-fold in assay buffer supplemented with ThT and incubated at 37°C for 45 min. The final concentration of peptides and ThT was 20 and 25 µM, respectively. (F, G) The bar diagram shows the mean ThT fluorescence and error bars the corresponding SD (n = 3). Statistical significance was assessed by ordinary one-way ANOVA (n.s.: p > 0.05, ****p ≤ 0.0001).
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Figure 4—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig4-data1-v1.zip
Figure 4—figure supplement 1
Investigation of the effect of the Myc-tag.
(A) BN-PAGE of p53 isoforms with either N- or C-terminal Myc-tag. H1299 cells were transiently transfected with the indicated Myc-tagged proteins. Cell lysates were subsequently analyzed by BN-PAGE and SDS-PAGE (shown in (B)) followed by WB using an α-Myc antibody for detection. High molecular weight species corresponding to aggregates are marked by ‘a’.
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Figure 4—figure supplement 1—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 2
Solubility of p53 isoforms.
(A) Representative WB of the solubility assay from Figure 4F. Transfected proteins were detected using an α-Myc antibody. Vinculin served as a control for the proper separation of the soluble (S) and insoluble (I) fraction.
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Figure 4—figure supplement 2—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig4-figsupp2-data1-v1.zip
Figure 4—figure supplement 3
Chaperone interaction of p53 isoforms.
(A) Solubility assay of p53 isoforms in new vector system (p5RPU.myc). H1299 cells were transiently transfected with the indicated Myc-tagged proteins and lysed in the respective buffers. Lysates were separated by centrifugation with the soluble components being in the supernatant (S) and the insoluble in the pellet (I). Samples were subsequently analyzed by WB using an α-Myc antibody. Vinculin served as loading control. (B) Solubility assay of Δ40p53α in the presence either DMSO or different inhibitors for HSP70 (JG-98, YM-1, VER-155008) or HSP90 (17-AAG) showing that these inhibitors have virtually no effect on the solubility of this isoform. (C) and (D) Same assay as in (E) but with the isoforms ΔN133p53α (F) and Δ133p53β (G) demonstrating that the use of chaperone inhibitors increases the insoluble fraction.
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Figure 4—figure supplement 3—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig4-figsupp3-data1-v1.zip
Figure 5 with 2 supplements
Chaperones.
(A) Co-immunoprecipitation of p53 isoforms and cancer-related mutants with endogenous HSC/HSP70. H1299 cells were transiently transfected with either empty vector or the indicated N-terminally Myc-tagged p53 variants. HSC/HSP70 was immunoprecipitated (IP) with an αHSC/HSP70 antibody. Input and IP samples were subsequently analysed by WB using αHSC/HSP70 and α-Myc antibody to detect HSC/HSP70 and p53 variants, respectively. A light and dark exposure of the IP samples detected with α-Myc antibody is shown. Vinculin served as a loading control for the input samples. (B) Luciferase reporter assay of p53 isoforms and mutants as well as the indicated Scarlet fusion proteins on the HSP70 promoter. H1299 cells were transiently transfected with the respective luciferase reporter plasmids and the N-terminally Myc-tagged proteins. (B) p53 (331) contains only the C-terminal part common to p53α, p53β, and p53γ. The fluorescent protein Scarlet was fused with the C-termini of p53 isoforms. Scarlet alone served as a negative control. (C) Luciferase reporter assay of p53 isoforms and mutants as well as the indicated Scarlet fusion proteins on the heat-shock element (HSE) promoter (containing three repeats of heat shock element). H1299 cells were transiently transfected with the respective luciferase reporter plasmids and the N-terminally Myc-tagged proteins. (C) p53 (331) contains only the C-terminal part common to p53α, p53β, and p53γ. The fluorescent protein Scarlet was fused with the C-termini of p53 isoforms. Scarlet alone served as a negative control. (B, C) The bar diagram shows the mean fold induction relative to the empty vector control and error bars the corresponding SD (n = 3). 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). (D) Chaperones and other proteins associated with binding un-/misfolded proteins are shown, which are significantly enriched in the mass spectrometry analysis of p53 isoforms. The red nods represent the identified proteins assigned by the grey lines to the p53 isoforms they were enriched for. Significant hits were determined by setting the parameters: log2 enrichment greater than or equal to 0.5 and p-value less than 0.05 and proteins were filtered for the keywords (‘chaperone’, ‘unfolded protein binding’, and ‘misfolded protein binding’) in the GOBP (Gene Ontology Biological Process) and GOMF (Gene Ontology Molecular Function) terms. The plot was generated using DiVenn (v2.0). (E) Results from the second mass spectrometry experiment, focused on the quantification of p53 peptide precursors. As the biotin ligase was directly fused to p53, p53 peptides are quantified for all samples (log2-transformed protein LFQ intensity). Negative controls represent data from unbiotinylated lysates (no biotin added). (F) Peptides from the DNA-binding domains spanning residues 249–267 were quantified for all samples (log2-scaled precursor intensity), consistent with this peptide being part of all investigated isoforms. A peptide spanning amino acids 102–110 was absent or beyond the detection limit in the Δ133p53β sample but quantified in all other samples. (G) Peptides originating from the oligomerization domain were reliably quantified in samples of isoforms containing a full-length oligomerization domain (log2-scaled precursor intensity), but not in samples of isoforms containing the β- and ɣ-C-termini (except for one low intense TAp53β precursor ion).
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Figure 5—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig5-data1-v1.zip
Figure 5—figure supplement 1
Chaperones.
(A) Co-IP of p53 isoforms and cancer-related mutants. H1299 cells were transiently transfected with N-terminally Myc-tagged p53 variants. p53 was immunoprecipitated (IP) with either α-mouse IgG or α-HSP70 antibody. Input and IP samples were subsequently analyzed by WB using an α-Myc antibody. For the relative Co-IP efficiency each IP signal was normalized to the input signal. (B) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 5B were determined by WB using an α-Myc antibody. Vinculin served as loading control. p53 (331) contains only the C-terminal part common to p53α, p53β and p53γ. (C) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 5B were determined by WB using an α-Myc antibody. Vinculin served as loading control. p53β CT: aa 332-341; p53γ CT: aa 332-346; p53γ CT mut: aa 332-346 with Y327A L330A L334A L336A; p53ψ CT: aa 223-243.
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Figure 5—figure supplement 1—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig5-figsupp1-data1-v1.zip
Figure 5—figure supplement 2
Mass spectrometry based proteomics.
(A) Barplot (left) shows the number of quantified protein groups in each individual replicate of each experimental condition. Boxplot (right) shows the overall protein intensity (log2-transformed protein LFQ intensity) proteins in each individual replicate of each experimental condition. Replicates within conditions share similar number of protein group IDs and intensity levels. Notably, in all controls (grey), less proteins were identified overall (left) and quantified ones exhibit lower overall intensity values (right) compared to the turboID samples (coloured). (B) Scatterplot shows principal component analysis of all quantified proteins (log2-transformed protein LFQ intensity, invalid values omitted) for each replicate of each experimental condition. (C) Heatmap shows the hierarchical clustering of each replicate according to similarity of their overall quantified proteins using their Pearson correlation coefficients (based on log2-transformed protein LFQ intensity, two-sided). Both unsupervised approaches (B,C) yield distinct clusters for samples of the same condition, underscoring their preparational reproducibility, and show clear segregation of experimental conditions (coloured) from the control group (grey).
Figure 6 with 1 supplement
Inactivation of p53 family members.
(A) Luciferase reporter assay of TAp53α in combination with p53 isoforms and cancer-related mutants on the pBDS-2 reporter. H1299 cells were transiently transfected with the respective luciferase reporter plasmids, the N-terminally Flag-tagged TAp53α alone or together with the N-terminally Myc-tagged p53 variants. Luciferase reporter assay of TAp73α (B), TAp73β (C), or TAp63γ (D) in combination with p53 and p63 isoforms and cancer-related mutants on the pBDS-2 reporter. H1299 cells were transiently transfected with the respective luciferase reporter plasmids, the N-terminally HA-tagged TAp73α (B), TAp73β (C), or TAp63γ (D) alone, empty vector or together with the N-terminally Myc-tagged p53/p63 variants. (A–D) The bar diagram shows the mean fold induction relative to the empty vector control and error bars the corresponding SD (n = 3). 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). (E) Luciferase reporter assay of TAp63γ in combination with the ∆40p53α isoforms and cancer-related mutants on the pBDS-2 reporter. H1299 cells were transiently transfected with the respective luciferase reporter plasmids, the N-terminally Myc-tagged TAp63γ alone or together with the N-terminally Myc-tagged ∆40p53α variants. (F) Conformation-specific immunoprecipitation (Conf-IP) of p53, p63, or p73 isoforms and p53 variant (∆40p53α, ∆40p53β, ∆133p53α, and ∆133p53β). H1299 cells were transiently transfected with N-terminally HA-tagged p53, p63, or p73 isoforms and N-terminally Myc-tagged p53 variants (∆40p53α, ∆40p53β, ∆133p53α, and ∆133p53β). p53, p63, or p73 isoforms were immunoprecipitated (IP) with α-HA. p53α isoforms mostly hetero-tetramerize with different p53α isoforms, but co-aggregate with remaining p53 isoforms. p63 and p73 isoforms interact with p53 isoforms by co-aggregation. Input and IP samples were subsequently analysed by WB using αMyc antibody.
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Figure 6—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig6-data1-v1.zip
Figure 6—figure supplement 1
Inactivation of p53 family members.
(A) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 6A were determined by WB using an α-Myc and α-Flag antibodies. Vinculin served as loading control. (B) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 6B were determined by WB using an α-Myc and α-Flag antibodies. Vinculin served as loading control. (C) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 6C were determined by WB using an α-Myc and α-Flag antibodies. Vinculin served as loading control. (D) Expression levels of the transiently transfected proteins in the luciferase reporter assay from Figure 6D were determined by WB using an α-Myc and α-Flag antibodies. Vinculin served as loading control.
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Figure 6—figure supplement 1—source data 1
Uncropped Western blots.
- https://cdn.elifesciences.org/articles/103537/elife-103537-fig6-figsupp1-data1-v1.zip
Figure 7
Mechanisms of inactivation within the p53 family.
p53 isoforms have the potential to inactivate p53 family members via different mechanisms. (A) Either p53α isoforms form inactive hetero-tetramers (and/or aggregate) with wtp53 (Δ40p53α, Δ133p53α, and Δ160p53α) and therefore inhibit wtp53 or inactive wtp53 by promoter squelching (Δ40p53α). (B) Furthermore, Δ40p53α isoform can as well inactivate tetrameric p63/p73 via promoter squelching. DNA-binding domain (DBD)-truncated aggregating p53 isoforms can co-aggregate with tetrameric p63α and p73α isoforms, but not with the closed TAp63α dimer.
Author response image 1
Predicted Chaperon binding sites using the LIMBO prediction tool.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | H1299 | ATCC | CRL-5803 | |
| Cell line (human) | SAOS | ATCC | HTB-85 | |
| Cell line (human) | U-2OS Flp-In T-Rex | Gift from C. Behrends | Generated according to the protocol of Thermo Fisher Scientific using the Flp-in T-Rex core lot (cat number K650001) | |
| Antibody | anti-Myc | Clone 4A6, Merck KGaA | 05-724 | 1:1000 |
| Antibody | anti-HA goat polyclonal | Bethyl Laboratories | A190-107A | 1:1000 |
| Antibody | anti-p53 (DO-1) | Santa Cruz Biotechnologies | sc-126 | 1:1000 |
| Antibody | anti-p53 (pAb240) | Santa Cruz Biotechnologies | sc-99 | 1:1000 |
| Antibody | anti-Vinculin | Santa Cruz Biotechnology Clone 7F9 | sc-73614 | 1:1000 |
| Antibody | anti-GST-HRP | GE Healthcare Life Sciences | RPN1236 | 1:10,000 |
| Antibody | anti-Streptavidin-HRP | Sigma-Aldrich Chemie GmbH | S2438 | 1:10,000 |
| Antibody | anti-Flag | Sigma-Aldrich Chemie GmbH Clone M2 | F3165 | 1:1000 |
| Antibody | anti-HSP70/HSC70 | Santa Cruz Biotechnology Clone W27 | sc-24 | 1:1000 |
| Antibody | Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-035-062 | 1:2000 |
| Antibody | Peroxidase AffiniPure Donkey Anti-Goat IgG (H+L) | Jackson ImmunoResearch | 705-035-003 | 1:2000 |
| Recombinant DNA reagent | pcDNA3.Myc | Thermo Fisher Scientific | Derivative of pcDNA3.1(+) (cat number V79020) | |
| Recombinant DNA reagent | p5RPU.Myc | Thermo Fisher Scientific | Modified from pcDNA3.Myc | |
| Recombinant DNA reagent | pcDNA3.HA | Thermo Fisher Scientific | Derivative of pcDNA3.1(+) (cat number V79020) | |
| Recombinant DNA reagent | pcDNA3.Flag | Thermo Fisher Scientific | Derivative of pcDNA3.1(+) (cat number V79020) | |
| Recombinant DNA reagent | pGL3-Basic vector | Promega | ||
| Recombinant DNA reagent | pRL-CMV | Promega | E2261 | |
| Recombinant DNA reagent | pBV-Luc BDS-2 3x WT | Addgene | Plasmid #16515 | |
| Recombinant DNA reagent | pGL2 basic vector with Mdm2 promoter | Promega | Derivative of pGL2 (GenBank X65323) | |
| Recombinant DNA reagent | pGL3 basic vector with p21 promoter | Promega | Derivative of pGL3 (GenBank U47295.2) | |
| Recombinant DNA reagent | pGL3 basic vector with HSP70 promoter | Promega | Derivative of pGL3 (GenBank U47295.2) | |
| Recombinant DNA reagent | pGL3 basic vector with HSP70/3x heat-shock elements (HSEs) | Promega | Derivative of pGL3 (GenBank U47295.2) | |
| Recombinant DNA reagent | pcDNA5/FRT/TO | Thermo Fisher Scientific | V652020 | |
| Recombinant DNA reagent | pOG44 | Thermo Fisher Scientific | V600520 | |
| Recombinant DNA reagent | pGEX-6P-2 | Cytiva | 28954650 | |
| Recombinant DNA reagent | p3036 GST-HPV-16 E6 | Addgene | Plasmid #10849 | |
| Recombinant DNA reagent | V5-TurboID-NES_pCDNA3 | Addgene | Plasmid #107169 | |
| Commercial assay or kit | Dual-Glo Luciferase Assay System | Promega | E2920 | |
| Commercial assay or kit | TnT Coupled Reticulocyte Lysate System | Promega | L4610 | |
| Commercial assay or kit | Trans-Blot Turbo RTA Midi PVDF Transfer Kit | Bio-Rad | #1704275 | |
| Chemical compound, drug | Lipofectamine 2000 transfection reagent | Thermo Fisher Scientific | 11668027 | |
| Chemical compound, drug | Pierce magnetic Streptavidin Beads | Thermo Fisher Scientific | 88817 | |
| Chemical compound, drug | Protein G Dynabeads | Thermo Fisher Scientific | 10003D | |
| Chemical compound, drug | Amersham ECL Prime WB Detection Reagent | Cytiva | RPN2232 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/103537/elife-103537-mdarchecklist1-v1.pdf
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Supplementary file 1
Core protein hits for p53 isoforms associated with various chaperones in-vivo.
- https://cdn.elifesciences.org/articles/103537/elife-103537-supp1-v1.pdf
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Source code 1
Code used to analyse the proteomics data.
- https://cdn.elifesciences.org/articles/103537/elife-103537-code1-v1.zip
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p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners
eLife 13:RP103537.
https://doi.org/10.7554/eLife.103537.3
