Journal: Journal of Cellular and Molecular Medicine

Article Title: Ozone therapy promotes the differentiation of basal keratinocytes via increasing Tp63‐mediated transcription of KRT10 to improve psoriasis

doi: 10.1111/jcmm.15160

Figure Lengend Snippet: Tp63 is involved in ozone‐mediated KC cell differentiation in vitro. A and B, the protein levels of Tp63 were examined in blank, IMQ, IMQ + Vehicle and IMQ + Ozone groups by IHC staining (A) and immunoblotting (B). C and D, The protein levels of Tp63 were examined in normal skin samples and psoriasis lesions with or without the ozone therapy by IHC staining (C) and immunoblotting (D). E and F, KCs were stimulated with IL‐22 and examined for the mRNA expression (F) and protein levels of Tp63 (E) with or without ozone treatment. G, ChIP assays were performed in 293T cells with anti‐Tp63 antibody to validate the binding of Tp63 to KRT10 promoter region. H, Luciferase reporter assays were performed in 293T cells to validate the effects of Tp63 on KRT10 transcription activity. I and J, KCs were transfected with si‐Tp63 in the presence or absence of ozone treatment and examined for the mRNA expression and protein levels of Tp63 and KRT10 by real‐time PCR (I) and immunoblotting (J). * P < .05, ** P < .01, compared to the control group; # P < .05, ## P < .01, compared to the non‐treated group

Article Snippet: To evaluate the protein contents of IL‐17, IL‐22, Tp63, KRT6 and KRT10, tissue sections were fixed in acetone for 10 minutes at −20°C, permeabilized with 0.2% triton (Sigma) for 10 minutes at room temperature, incubated with a blocking solution (3.75% BSA/5% goat serum, Zymed) for 30 minutes and incubated for 2 hours with IL‐17 (26163‐1‐AP; Proteintech), IL‐22(ab203211; Abcam), Tp63 (PA2056; Bosterbio), KRT6 (10590‐1‐AP; Proteintech) or KRT10 (sc‐53252; Santa Cruz).

Techniques: Cell Differentiation, In Vitro, Immunohistochemistry, Western Blot, Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ozone therapy promotes the differentiation of basal keratinocytes via increasing Tp63‐mediated transcription of KRT10 to improve psoriasis

doi: 10.1111/jcmm.15160

Figure Lengend Snippet: Expression and correlation of Tp63 and KRT10 in tissue samples in vivo. A and B, The expression of Tp63 and KRT10 in psoriasis lesion tissues with or without ozone treatment were determined by real‐time PCR. C, The correlation of Tp63 and KRT10 expression in tissue samples was analysed by Pearson's correlation analysis

Article Snippet: To evaluate the protein contents of IL‐17, IL‐22, Tp63, KRT6 and KRT10, tissue sections were fixed in acetone for 10 minutes at −20°C, permeabilized with 0.2% triton (Sigma) for 10 minutes at room temperature, incubated with a blocking solution (3.75% BSA/5% goat serum, Zymed) for 30 minutes and incubated for 2 hours with IL‐17 (26163‐1‐AP; Proteintech), IL‐22(ab203211; Abcam), Tp63 (PA2056; Bosterbio), KRT6 (10590‐1‐AP; Proteintech) or KRT10 (sc‐53252; Santa Cruz).

Techniques: Expressing, In Vivo, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

doi: 10.1371/journal.pone.0051426

Figure Lengend Snippet: ( A ) Overexpressed HSP70 restores transcriptional activity of TAp63α isoform inhibited by p53 R175H mutant in H1299 cells (left diagram), while TAp73α remains inhibited (right diagram). All data points were carried out in triplicate by means of Dual Luciferase Reporter Assay. ( B ) Overexpressed HSP70 molecular chaperone releases TAp63α isoform from the complex with p53 R175H mutant in H1299 cells, whereas maintains TAp73α in this interaction. 24h post-transfection cells were lysed and p53 protein was immunoprecipitated with anti-p53 antibody (DO-1). The immunoprecipitated protein complexes were subjected to Western blot analysis. ( C ) H1299 cells were transfected with plasmids encoding p53 R175H and p63 or p73 and treated with MG132 (2 µM) overnight. Labelling for specific antibodies showed that under such conditions p73 coaggregates with mutant p53 in the nucleus, while p63 remains diffused in the same compartment. Analysis performed in the ImageJ software with the JACoP plugin confirmed that the colocalization is high for p73 and p53 R175H, whereas p63 overlaps with mutant p53 only partially (diagram). Two colocalization factors were determined – Pearson’s coefficient (r) and Mander’s coefficients (M1 and M2) - using threshold value of 110 for images in p63 analysis (upper panels) and 95 for p73 (lower panels). ( D ) p63 and p73 protein solubility assay performed in H1299 cells cotransfected with plasmids encoding one of those two proteins parallel with p53 R175H and HSP70 revealed that p63 is absent in the most detergent resistant fraction, while a part of p73 pool remains aggregated and is present in the Laemmli sample buffer together with mutant p53 and HSP70. Detailed description of the method available in , , , , , , , , .

Article Snippet: The following antibodies for Western blotting, immunofluorescence and immunoprecipitation were used: p53 (DO-1, pAb 1620, pAb 240), p73 (rabbit polyclonal) and MDM2 (4B2) – all a kind gift from B. Vojtesek), HSP70 (SPA-812, Stressgen; 6B3, Cell Signalling), HSP40 (SPA-400, Stressgen), HSP90 (SPA-835, SPA-846, Stressgen), HA-tag (3F10, Roche Molecular Biochemicals), γ-tubulin (Abcam), p63 (4A4, Santa Cruz), amyloid oligomers (A11, Calbiochem).

Techniques: Activity Assay, Mutagenesis, Luciferase, Reporter Assay, Transfection, Immunoprecipitation, Western Blot, Software, Solubility

Journal: PLoS ONE

Article Title: Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

doi: 10.1371/journal.pone.0051426

Figure Lengend Snippet: Our data suggests that the pro-oncogenic, gain-of-function phenotype of mutant p53 is governed by HSP70 and MDM2 levels. Recurrent interaction of HSP70 with the p53 polypeptide, in an ATP dependent manner, causes transient exposure of its aggregate prone domain(s). Subsequent aggregation of mutant p53 is further augmented by the MDM2-p53 allosteric interaction. This dynamic, irreversible molecular process can sequester other tumour suppressors, such as p73, thus inhibiting their activity. Pro-oncogenic activities of mutant p53 can be manifested through other, non-sequestration based mechanisms as depicted (described previously ). We cannot exclude the possibility that molecular chaperones, including HSP70, are also involved in these processes.

Article Snippet: The following antibodies for Western blotting, immunofluorescence and immunoprecipitation were used: p53 (DO-1, pAb 1620, pAb 240), p73 (rabbit polyclonal) and MDM2 (4B2) – all a kind gift from B. Vojtesek), HSP70 (SPA-812, Stressgen; 6B3, Cell Signalling), HSP40 (SPA-400, Stressgen), HSP90 (SPA-835, SPA-846, Stressgen), HA-tag (3F10, Roche Molecular Biochemicals), γ-tubulin (Abcam), p63 (4A4, Santa Cruz), amyloid oligomers (A11, Calbiochem).

Techniques: Mutagenesis, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

doi: 10.1038/cdd.2016.83

Figure Lengend Snippet: Co-expression and hetero-tetramerization of endogenous p63 and p73. (a) IHC staining of P5 mouse skin sections for p63 and p73. Sections include the epidermis and dermis, latter harboring the hair follicles. Stained epidermis and hair follicle structures are shown in detail. The black dashed line marks the transition between epidermis and dermis. Scale bar: 25 μm. (b) Representative IF staining of skin from a new-born (P5) mouse for p63 and p73. Both, in the epidermis (upper panel) and hair follicle (lower panel), p73 (green) colocalizes with p63 (red) in a subset of K14-positive cells (cyan). In the epidermis, p63 and K14-positive cells are located in the basal layer at the boundary between epidermis and dermis (white dashed lines, upper panel)). In the anagen hair follicle (white dashed lines, lower panel), p63 is expressed by the cells of the matrix (M) and the outer rood sheet (ORS). K14-positive cells from the outer layer of the ORS show colocalization of p63 and p73. The structure and expression pattern of the interfollicular epidermis (IFE) is equal to the regular epidermis. White boxes mark the magnified area. Scale bar: 25 μm. Expression level of p63, p73 and K10 during keratinocyte differentiation. Differentiation of keratinocytes was induced by supplementing the culture medium with high calcium concentrations. p63 and p73 levels were detected via western blot on consecutive days. p73 initially shows a very low basal expression. At day 2 after induction, the protein level sharply increases and is gradually lost again after day 3. p63 levels are initially on a high level, but the expression gradually decreases after day 2 (note that for the western blot in panel c a lower amount of the p63 antibody was used than for the p63 western blot shown in d). Uninduced control cells were blotted as well (‘-‘). K10 expression (red line in panel p63 symbolizing the expression profile) is detectable from day 3 on. (d) Differentiated and non-differentiated primary human keratinocyte lysate was immunoprecipitated with a p73-specific antibody and a p63-specific antibody was used for detection and vice versa. Keratinocyte differentiation was induced with high calcium concentrations (dx=day × after induction). Normal IgG was used as a control. IP, immunoprecipitation; WB, western blot

Article Snippet: After centrifugation, the supernatant was incubated with 2 μ g of anti-p63 (H-129, Santa Cruz Biotechnology), anti-p73 (ER-15, Merck Millipore), normal IgG (rabbit or mouse, Santa Cruz Biotechnology) or anti-p63 (D2K8X, CST) in a 1:800 dilution overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

doi: 10.1038/cdd.2016.83

Figure Lengend Snippet: Structure of the hetero-tetramer between the TDs of p63 and p73. (a) Sequence alignment of p63 and p73 TDs. The TDs form salt bridges across the central tetrameric interface. Here we introduced charge-swapped mutants of p63 TD (K377E) and p73 TD (E363K) that are designed to exclusively form hetero-tetramers. (b) p63 TD and p73 TD form several different hetero-tetrameric species when mixed. Here we show schematically that the designed p63 TD hetero and p73 TD hetero mutants exclusively form hetero-tetramers. (c) Section of [15N, 1H]-TROSY spectra of 15N-lysine-labeled p73 TD hetero (E363K) in absence (blue) or presence of an equimolar amount of p63 TD hetero (K377E) (red), showing K372. (d) Section of [15N, 1H]-TROSY spectra of 15N-lysine-labeled p63 TD hetero (K377E) in absence (blue) or presence of an equimolar amount of p73 TD hetero (E363K) (red), showing K379. (e) The hetero-tetramer of p63/p73 (PDB 2NB1) is composed of two chains of each protein. For clarity reasons, the flexible c-termini of the p63 chains after Q409 have been omitted. p63 chains (blue) and p73 chains (red) each form an independent dimer consisting of an N-terminal antiparallel β-sheet and one subsequent helix per monomer. These helices interact with the corresponding helices from the other dimer, thus forming the central tetramerization interface. In addition, the C-terminal helix 2 leans toward the β-strand from the opposing dimer. (f) View onto the p63 β-sheet as a surface model with helices 2 of p73 shown as explicit secondary structure element. The hydrophobic outward facing amino acids of the p63 chains are depicted as colored sticks (L361 in orange, Y363 in yellow, P365 in blue). The side chain of L396 of p73 (red, shown as sticks) penetrates into a hydrophobic pocket formed mainly by P365 of p63. L361 shows a small additional stabilization, however, the main effect can be attributed to P365 (Supplementary Figure S4). NOE restraints, which could be detected in this region, are shown as green lines

Article Snippet: After centrifugation, the supernatant was incubated with 2 μ g of anti-p63 (H-129, Santa Cruz Biotechnology), anti-p73 (ER-15, Merck Millipore), normal IgG (rabbit or mouse, Santa Cruz Biotechnology) or anti-p63 (D2K8X, CST) in a 1:800 dilution overnight at 4 °C.

Techniques: Sequencing, Labeling

Journal: Cell Death and Differentiation

Article Title: Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

doi: 10.1038/cdd.2016.83

Figure Lengend Snippet: Hetero-tetramerization between the TDs of p63 and p73. All mutations described in panels c–f were designed based on the NMR solution structure of the hetero-TD. The mutations were carried out in wild-type proteins, therefore they do not carry the hetero-stabilizing mutations. (a and b) Sections of [15N,1H]-BEST-TROSY spectra of selectively lysine-labeled wild-type p63 (a) and wild-type p73 (b) showing K377 or K372, respectively. A total of eight different resonances can be detected after mixing each protein with equal amounts of unlabeled p73 or p63, respectively, and subsequent incubation for 24 h at 37 °C. Peaks corresponding to the homo-tetramers in a and b are overlaid in green and blue, respectively. Peaks corresponding to the homo-tetrameric population, as well as the hetero-tetramer of homo-dimers, are marked in each panel. The mayor single population in both cases is the hetero-tetramer of homo-dimers. (c and d) Sections of [15N,1H]-BEST-TROSY spectra of p63 and mutant p73 (L395A, L396A), prepared analogous to a and b. In contrast to a and b, the magnitude of hetero-tetramers of homo-dimers is drastically reduced. (e and f) Sections of [15N,1H]-BEST-TROSY spectra of mutant p63 (L361T, P365Q) and p73 prepared analogous to a and b. L361 and P365 are the only amino acids of the outward facing leaf of the p63 TD, which are different to p73. Both were mutated to the corresponding amino acids of p73, which also leads to a drastic reduction of the formation of hetero-tetramers of homo-dimers. (g) The intensities of all visible populations in each spectrum can be used to calculate the percentage of each possible combination of homo and hetero-tetramers in solution. For the wild-type protein (red bars), the hetero-tetramer of homo-dimers is the most stable and therefore most prominent population, whereas for both double mutants (orange and blue bars) the percentage of this population is drastically reduced, almost reaching the statistically expected values (black bars)

Article Snippet: After centrifugation, the supernatant was incubated with 2 μ g of anti-p63 (H-129, Santa Cruz Biotechnology), anti-p73 (ER-15, Merck Millipore), normal IgG (rabbit or mouse, Santa Cruz Biotechnology) or anti-p63 (D2K8X, CST) in a 1:800 dilution overnight at 4 °C.

Techniques: Labeling, Incubation, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

doi: 10.1038/cdd.2016.83

Figure Lengend Snippet: Analysis of the p73 homo mutation. (a) SEC profile of p73 TD and p73 TD homo measured on a Superdex 75 10/300. (b) Sections of [15N, 1H]-TROSY spectra of 15N-lysine-labeled p73 TD homo in absence (blue) or presence (red) of an equimolar amount of p63 TD (left) or p53 OD (right) measured after incubation for one day at 37 °C. (c) SEC profiles of TAp73α (blue) and TAp73α homo (E363R, K370E, E373R, R390D) (orange) produced in RRL. (d) Crystal structure of p73 TD homo (PDB 5HOC). Side chains of E363R, K370E, E373R and R390D are shown. Data collection and refinement statistics are provided in Supplementary Table 2. (e) Structural alignment of p73 TD homo with p63 TD (PDB 4A9Z), showing a hypothetical and non-favored hetero-tetramer of p63 TD and p73 TD homo. Electrostatic repulsions between negatively charged side chains (encircled in red) or positively charged side chains (encircled in blue) explain why p73 TD homo forms exclusively homo-tetramers. (f) Close-up view of the central tetrameric interface of p73 TD homo. 2Fo-Fc density maps are shown for the side chains of E363R, K370E, E373R and R390D

Article Snippet: After centrifugation, the supernatant was incubated with 2 μ g of anti-p63 (H-129, Santa Cruz Biotechnology), anti-p73 (ER-15, Merck Millipore), normal IgG (rabbit or mouse, Santa Cruz Biotechnology) or anti-p63 (D2K8X, CST) in a 1:800 dilution overnight at 4 °C.

Techniques: Mutagenesis, Labeling, Incubation, Produced

Journal: Cell Death and Differentiation

Article Title: Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

doi: 10.1038/cdd.2016.83

Figure Lengend Snippet: Introduction of homo mutations in p63 or p73 abrogates interaction in cells. Wild-type ΔNp63α, ΔNp63α hetero, ΔNp63α homo and wild-type TAp73β, TAp73β hetero or TAp73β homo were co-transfected in SK-N-AS cells. Immunoprecipitation (IP) of p63 (a and b) or p73 (c and d) was performed with subsequent analysis by western blot with a p73-specific antibody or a p63-specific antibody, respectively. Western blots were quantified using ImageJ and co-IP efficiency of wild-type proteins was set to 1 b and d

Article Snippet: After centrifugation, the supernatant was incubated with 2 μ g of anti-p63 (H-129, Santa Cruz Biotechnology), anti-p73 (ER-15, Merck Millipore), normal IgG (rabbit or mouse, Santa Cruz Biotechnology) or anti-p63 (D2K8X, CST) in a 1:800 dilution overnight at 4 °C.

Techniques: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay