Densitometry was done with imagej software

The exchange of monomers within a p53 dimer was indeed found to be ultra-slow in purified p53 proteins in vitro, and p53 monomer and hetero-dimer concentrations were found to be limited in MCF7 cells - using fluorescence correlation spectroscopy and protein-fragment complementation assay.

This effect can be partially attributed to a co-translational dimerization of p53, discovered earlier using an in vitro translation system, which implies that usually mutant p53 enters p53 tetramers as a homodimer вЂ“ resulting in a limited inactivation of the DNA binding by wt p53, which could be partially retained in the wt p53 homo-dimer. measured the functional efficiency of the dominant-negative effect to be surprisingly low вЂ“ as excess of mutant p53 protein was required to inactivate the wt p53 activity. Oligomerization of wt p53 also represents one of its functional weaknesses, as hot-spot mutant p53 variants were observed to inactivate wt p53 by hetero-oligomerization via a dominant-negative (DN) mechanism. Numerous studies - structural and functional - have demonstrated that a tetrameric form of wt p53 is optimal for its effective binding to a target promoter DNA and its function as the tumor-suppressive transcription factor. The majority of the TP53 mutations result in single residue changes in p53 proteins, most of which вЂ“ including the common вЂњhot-spotвЂќ mutations вЂ“ inactivate DNA-binding and tumor suppressor functions of wild-type (wt) p53, endowing mutant p53 proteins with transforming, gain-of-function (GOF) oncogenic properties. TP53 gene, encoding p53 protein, is the most frequently mutated locus overall in human neoplasias. These results suggest that the frequent mutant p53 accumulation in human tumor cells does not only directly strengthen its gain-of-function activity, but also protects the oncogenic p53 mutants from the functional dominance of wt p53.

Indeed, we show that at similar wt:mutant p53 concentrations in cells вЂ“ the mutant p53 gain-of-function stimulation of gene transcription and cell migration is more efficiently inhibited than the wt p53вЂ™s tumor-suppressive transactivation and suppression of cell migration. The biased p53 oligomerization helps to interpret earlier reports of a low efficiency of the wt p53 inactivation via the dominant-negative effect, while it also implies that the вЂњdominant-positiveвЂќ effect may be more pronounced. In this study we determine the oligomerization efficiency of wt and mutant p53 in living cells using FRET-based assays and describe wt p53 to be more efficient than mutant p53 in entering p53 oligomers. Oligomerization of p53 allows for a direct interplay between wt and mutant p53 proteins if both are present in the same cells вЂ“ where a mutant p53вЂ™s dominant-negative effect known to inactivate wt p53, co-exists with an opposite mechanism вЂ“ a вЂњdominant-positiveвЂќ suppression of the mutant p53вЂ™s gain-of-function activity by wt p53. Single residue changes, caused by hot-spot mutations of the TP53 gene in human cancer, transform wild-type (wt) p53 tumor suppressor proteins into potent oncoproteins - with gain-of-function, tumor-promoting activity. Human p53 protein acts as a transcription factor predominantly in a tetrameric form. Received: FebruAccepted: JPublished: August 10, 2018 Dawid Walerych 1, Magdalena Pruszko 1, Lukasz Zyla 2, Michalina Wezyk 1, Katarzyna Gaweda-Walerych 1 and Alicja Zylicz 2ДЎMossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, PolandДўInternational Institute of Molecular and Cell Biology in Warsaw, 02-109 Warsaw, PolandДawid Walerych, email: p53 oligomerization FRET dominant-negative gain-of-function

Densitometry was done with imagej software