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Vandemoortele, G.

Publications and source records attributed to Vandemoortele, G..

2 recordsLinked to original sources

Proximity map for p53 unveils a strong link with PML Nuclear Bodies in HEK293 cells

The tumor suppressor p53 is among the most studied proteins in cancer, with mutations in nearly half of all tumors. Even when TP53 is intact, its regulatory pathways are often disrupted, underscoring its central role in tumorigenesis. To explore how cellular context shapes the subcellular environment of p53, we used BioID proximity labeling in HEK293 cells. These experiments revealed a striking enrichment of proteins involved in SUMOylation and proteins that reside in PML nuclear bodies. A strong enrichment of the adenoviral E1B protein, originally used for transformation of this cell line, may explain this striking observation, and can shed light on the remarkable difference with the proximity map of p53 in the HCT116 colon carcinoma cell line. Strong enrichment of the mediator complex in HCT116, related to active p53-dependent transcription, is missing in 293 cells and can explain the lack of p53 transcriptional activity in these cells. This study emphasizes how cellular transformation can affect the proximal proteome of a key protein in cell cycle control, DNA repair, apoptosis, senescence and metabolism, and confirms previously reported inhibition of p53-dependent transcription by sequestering the protein in PML nuclear bodies.

systems biology↗

A well-controlled BioID design for endogenous bait proteins.

The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate overexpression of bait proteins. Control cell lines are typically non-engineered cells or engineered clones implying a considerable risk for artefacts because of clonal variation. Genome engineering can also transform BioID, a proximity labelling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bi-cistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A auto-cleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over classical ectopic expression of p53-BirA*, and we provide a first well-controlled view on the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC=\"FIGDIR/small/427807v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@404da1org.highwire.dtl.DTLVardef@1a8af36org.highwire.dtl.DTLVardef@18741a6org.highwire.dtl.DTLVardef@e73ab3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗