Project description:The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, we analyzed pancreatic cancer predisposition in mice expressing p53 transactivation domain (TAD) mutants. Surprisingly, we observed that p53 TAD2 mutant behaves as a “super-tumor suppressor”, with an enhanced capacity to suppress pancreatic cancer and to activate a subset of novel p53 target genes. One such gene, Ptpn14, is a direct p53-inducible gene encoding a negative regulator of the Yap oncoprotein. To determine the effects of p53 deficiency on Yap target gene expression in pre-malignant mouse pancreatic intraepithelial neoplasia (PanIN) lesions, we sorted the CD133-positive ductal epithelial cells that make up the PanINs and used these cells for RNA-sequencing analysis. This analysis revealed that a Yap signature is induced upon p53 deficiency, suggesting that p53 loss promotes the induction of a Yap transcriptional program.
Project description:It is a TMT10Plex proteomics dataset used for relative protein and phosphoprotein quantification in the manuscript entitled "Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function". It contains both non-phosphorylated (used for identification and quantification of total proteomes) and phospho enriched (used for identification and quantification of phosphoproteins) dataset.
Project description:The tumor suppressor protein p53 is a transcription factor that is referred to as the “guardian of the genome” and plays an important role in cancer development. P53 is active as a tetramer; the S100β homodimer binds to the intrinsically disordered C-terminus of p53, affecting its transcriptional activity. The p53/S100β complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100β exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100β complexes by electrospray ionization mass spectrometry (ESI-MS), cross-linking mass spectrometry (XL-MS), and surface plasmon resonance (SPR). For this, wild-type p53 and single amino acid variants, representing different oligomeric states of p53 (tetrameric wild-type, dimeric L344A variant, and monomeric L344P variant) were individually investigated regarding their binding behavior towards S100β. The stoichiometry of the different p53/S100β complexes were determined by ESI-MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100β dimer. In addition, XL-MS revealed the topologies of the p53/S100β complexes to be independent of p53’s oligomeric state. With SPR, the thermodynamic parameters were determined for S100β binding to tetrameric, dimeric or monomeric p53 variants. Our data prove that the S100β homodimer binds to different oligomeric states of p53 with identical stoichiometries and similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100β interaction.
Project description:The tetrameric tumor suppressor p53 represents a great challenge for 3D structural analysis due to its high degree of intrinsic disorder (ca. 40%). We developed and applied an integrative structural biology approach combining complementary techniques of structural mass spectrometry (MS), namely cross-linking mass spectrometry (XL-MS), protein footprinting, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), with advanced protein structure prediction approaches to gain insights into the disordered C-terminal region of p53. Additionally, we evaluate possible differences in p53 regarding solvent accessibility and topology upon DNA binding. Our quantitative XL-MS and lysine labeling data show no major conformational differences in p53 between DNA-bound and DNA-free states. Integration of experimental data generate p53 models for p53’s intrinsically disordered regions (IDRs) that reflect substantial compaction of the molecule. Our models provide the most detailed description of the relationship between p53’s folded regions and IDRs that is available to date. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and IDRs.
Project description:Alteration of metabolism in cancer cells is a central aspect of the mechanisms that sustain aggressive traits. Aldo-keto reductase 1 B1 (AKR1B1) catalyzes the reduction of several aldehydes to alcohols consuming NADPH. Nevertheless, the ability of AKR1B1 to reduce different substrates renders difficult to comprehensively ascertain its biological role. Recent evidence has implicated AKR1B1 in cancer, however, the mechanisms underlying its pro-oncogenic function remain largely unknown. In this work, we report that AKR1B1 expression is controlled by the p53 tumor suppressor. We found that breast cancer patients bearing wild type TP53 have reduced AKR1B1 expression. In cancer cell lines, p53 reduced AKR1B1 mRNA and protein levels, and repressed promoter activity in luciferase assays. Furthermore, chromatin immunoprecipitation assays indicated that p53 is recruited to the AKR1B1 promoter. We also observed that AKR1B1 overexpression promoted metastasis in the 4T1 orthotopic model of triple negative breast cancer. Proteomic analysis of 4T1 cells overexpressing AKR1B1 showed that AKR1B1 exerts a marked effect on proteins related to metabolism, with particular impact on mitochondrial function. This work provides novel insights on the link between the p53 pathway and metabolism in cancer cells and contributes to characterizing the alterations associated to the pathologic role of AKR1B1.
Project description:Transcriptome analysis of protein mimetic amyloid inhibitor that potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function