The Δ133p53 and Δ160p53 proteins exhibit structural destabilization.

(A) Schematic domain structures of full-length (FL) p53 and its isoforms. TAD, transactivation domain; PRD, proline-rich domain; DBD, DNA-binding domain; NLS, nuclear localization sequence; OD, oligomerization domain; CTD, C-terminal domain. Δ133p53 lacks the entire TAD and approximately 20% of the DBD. Δ160p53 lacks the entire TAD and approximately 33% of the DBD. An aggregation region ILTIITL (residues 251-257) is indicated in the DBD domain. (B) Contact density map (C-α 8Å) of the core domain of FLp53 (PDB ID: 3KMD) using CWView88. The residues 94-132 and 133-159 were colored magenta and green, respectively. Higher densities are shown in red, and low densities are shown in blue. Among these contacts, four higher-density contacts were selected and shown in oval circles on the map. The corresponding secondary structures of the contacts between the deleted region (blue) and the region (red) in the loop-sheet-helix (LSH) motif or the β-sandwich are shown on the right. (C) Comparison analysis of surface hydrophobicity of the core domain in FLp53 (PDB ID: 3KMD) and its isoforms Δ133p53 and Δ160p53 by ChimeraX89, 90. The exposed hydrophobic residues in the aggregation region ILTIITL (residues 251-257) are labeled. The color scale of hydrophobicity is indicated with the maximum and minimum values in dark cyan and dark goldenrod, respectively. The β-hairpin S2–S2′ was shown in an oval circle.

Complex formation among FLp53 and its isoforms Δ133p53 and Δ160p53.

(A) Co-immunoprecipitation of FLp53-FLAG, Δ133p53-FLAG, and Δ160p53-FLAG along with the FLp53-V5, Δ133p53-V5, and Δ160p53-V5 proteins in H1299 cells. The complexes were isolated with anti-FLAG antibody-coupled magnetic beads. Western blot analysis was done with the anti-FLAG and anti-V5 antibodies. The merged image corresponds to the overlay between the FLAG and V5 antibody signals. Input corresponds to 5% of the whole cell lysate. (B) A summary of FLp53, Δ133p53, and Δ160p53 oligomerization based on data shown in panel A.

Δ133p53 and Δ160p53 negatively influence FLp53’s DNA-binding activity.

(A) Schematic representation of the promoters of p53 target genes (p21, MDM2, BAX, and PUMA). An arrow denotes the transcription start site (TSS). The DNA sequences of p53 response elements (REs) are shown, with uppercase and lowercase letters indicating matched and mismatched bases, respectively, in relation to the canonical p53 RE sequence (RRRCWWGYYY). Paired arrows highlight the regions subjected to quantitative PCR (qPCR) amplification. Specifically, the distal (5’) p53 RE within the p21 gene promoter was analyzed. (B) Relative DNA-binding of the FLp53-FLAG, Δ133p53-FLAG, and Δ160p53-FLAG proteins to p53 target genes (p21, MDM2, BAX, and PUMA) in H1299 cells. (C) Relative DNA-binding of the FLp53-FLAG protein to the p53-target gene promoters in the presence of the V5-tagged protein Δ133p53 or Δ160p53 at a 1: 1 ratio. ChIP-qPCR assay data are shown as relative enrichment of promoter sequences of the target genes after normalization to the control, pcDNA3.1 plasmid transfected cells. Data are represented as the mean of technical triplicates ± standard deviation (SD), **P < 0.01 (Student’s t-test).

Impact of Δ133p53 and Δ160p53 on FLp53-activated transactivation.

(A-D) H1299 cells were transfected with luciferase reporter plasmids driven by p21 (A), MDM2 (B), BAX (C), and PUMA (D) promoters, along with vectors expressing FLp53, Δ133p53, or Δ160p53. To evaluate the influence of the isoforms on FLp53’s transactivation capability, co-expression was performed at ratios of 1:1, 1:5, and 1:10 relative to FLp53. Basal promoter activity was determined by transfecting cells with the empty vector, pcDNA3.1. Relative promoter activity is shown after normalization to the pcDNA3.1-treated sample activity. Data represent mean values ± standard deviation (SD) (n=3). *P < 0.05; **P < 0.01 (Student’s t-test). (E-H) The transcriptional activity of FLp53 on the p21 (E), MDM2 (F), BAX (G), and PUMA (H) promoters was inhibited by Δ133p53 and Δ160p53. Inhibition curve fitting was performed using the exponential function ExpDec1 in Origin 2018 software.

Subcellular distribution of the FLp53, Δ133p53 and Δ160p53 proteins.

(A) Western blot analysis of subcellular fractionation of H1299 cells transfected with the FLAG-tagged FLp53, Δ133p53, or Δ160p53 proteins. Representative immunoblots indicate the presence of these proteins in different cellular fractions. α-Tubulin (cytoplasmic marker) and Histone H3 (nuclear marker) were used to ensure fraction integrity. (B) Quantitative bar graph summarizing the cytoplasm-to-nucleus ratio of protein levels. FLAG-tagged protein levels were normalized to their respective fractionation markers: α-Tubulin for the cytoplasmic fraction and histone H3 for the nuclear fraction. The relative ratios of Δ133p53 and Δ160p53 protein levels are displayed, with the FLp53 cytoplasm-to-nucleus ratio set as 1. (C) Immunofluorescence analysis of H1299 cells expressing FLAG-tagged FLp53, Δ133p53 and Δ160p53. Cell nuclei are visualized with DAPI (blue). The punctate spots (white arrows) indicating protein aggregates are marked. Scale bar, 50 μm.

Induction of FLp53 aggregation by Δ133p53 and Δ160p53 isoforms.

(A-B) Western blot analysis of soluble cytoplasmic and nuclear subcellular fractions (A) and insoluble nuclear fraction (B). Biochemical fractionation of H1299 cells transfected with the FLAG-tagged FLp53 and V5-tagged Δ133p53 or Δ160p53 at a 1:5 ratio. The aggregated monomers of V5-tagged Δ133p53 and Δ160p53 (indicated by white arrows), as well as the higher molecular weight aggregates, are shown. This figure shows one representative experiment; n=3. (C) Quantitative bar graph summarizing the levels of the soluble FLp53-FLAG protein in the cytoplasmic and nuclear fractions. The FLp53-FLAG protein levels were normalized to the corresponding fractionation marker (α-Tubulin or histone H3). Relative accumulation of the FLp53-FLAG in Δ133p53-V5 or Δ160p53-V5 expressing cells is shown after considering the FLp53-FLAG protein expressing sample as 1. (D) Quantitative bar graph summarizing the levels of the FLp53-FLAG protein in the insoluble nuclear fraction relative to the histone H3. Data normalization as in panel C, data represent mean values ± standard deviation (SD). **P < 0.01 (Student’s t-test). (E) Immunofluorescence analysis of H1299 cells transfected with FLp53-FLAG alone or in combination with V5-tagged isoforms Δ133p53 and Δ160p53 expressing plasmids at a concentration ratio of 1:5. Cell nuclei were visualized with DAPI (blue). The co-aggregation of isoforms with FLp53 in the cytoplasm (white arrows) and nucleus (bright green arrows) are indicated. Scale bar, 50 μm.

Overexpression of Δ133p53 and Δ160p53 block pro-apoptotic activity of FLp53.

Caspase-3 and -7 activities were detected in H1299 cells co-transfected with plasmid expressing FLp53 or the isoforms Δ133p53 and Δ160p53 at a 1:5 ratio. Experiments were performed with the plasmids expressing FLAG-tagged and untagged FLp53, Δ133p53 and Δ160p53. The relative activities of caspase-3 and -7 were normalized to those of the control vector (transfected only with the pcDNA3.1 plasmid). Data represent mean values ± standard deviation (SD). **P < 0.01 (Student’s t-test).

(A) Schematic representation of the underlying mechanisms by which Δ133p53 and Δ160p53 exert dominant-negative effect on FLp53.

(A) When expressed alone, FLp53 demonstrates strong transcriptional activity, but when expressed together with the Δ133p53 or Δ160p53 isoforms, FLp53 activity gets impaired due to the dominant-negative effect exerted by the isoforms. The Δ133p53 or Δ160p53 isoforms either attenuate FLp53’s transcription activity by hetero-tetramerization in a ratio higher than 1:1, or abolish it completely by aggregation. Since Δ133p53 and Δ160p53 exhibit much higher propensity for aggregation than FLp53, they sequester FLp53 in the cytoplasmic aggregates, leading to reduction in the amount of functional FLp53 in the nucleus. Aggregation also limits the availability of these isoforms in the nucleus. Thus, co-aggregation is the primary reason for dominant negative effect of the Δ133p53 or Δ160p53 isoforms on FLp53. It is important to note that the numbers of tetramers and aggregates depicted do not represent the actual quantities present in the cytoplasm and nucleus. (B) The balance between the expression level of the FLp53 and its isoforms Δ133p53 and Δ160p53 regulates FLp53 activity. Increased levels of Δ133p53 and Δ160p53 relative to FLp53, lead to stronger dominant-negative effect due to co-aggregation and hetero-tetramerization, resulting in the inhibition of apoptosis. Increased levels of FLp53 compared to the isoforms Δ133p53 and Δ160p53 in cancer cells restore the function of FLp53, leading to the induction of apoptosis.