How Do Cancer-Related Mutations Affect the Oligomerisation State of the p53 Tetramerisation Domain?

Abstract

Tumour suppressor p53 plays a key role in the development of cancer and has therefore been widely studied in recent decades. While it is well known that p53 is biologically active as a tetramer, the tetramerisation mechanism is still not completely understood. p53 is mutated in nearly 50% of cancers, and mutations can alter the oligomeric state of the protein, having an impact on the biological function of the protein and on cell fate decisions. Here, we describe the effects of a number of representative cancer-related mutations on tetramerisation domain (TD) oligomerisation defining a peptide length that permits having a folded and structured domain, thus avoiding the effect of the flanking regions and the net charges at the N- and C-terminus. These peptides have been studied under different experimental conditions. We have applied a variety of techniques, including circular dichroism (CD), native mass spectrometry (MS) and high-field solution NMR. Native MS allows us to detect the native state of complexes maintaining the peptide complexes intact in the gas phase; the secondary and quaternary structures were analysed in solution by NMR, and the oligomeric forms were assigned by diffusion NMR experiments. A significant destabilising effect and a variable monomer population were observed for all the mutants studied.

Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aβ1‐40 by NMR

“…Starting from the measured D value the apparent MW of the oligomers was calculated by the combination of Stokes À Einstein equation [34,35] with the relationship between molar radius and formula weight. [33] Assuming a spherical model, dioxane molecule radius (r H diox = 0.212 nm) was used as standard to obtain estimates of the effective hydrodynamic radius of small oligomers (r obs H Þ, using the following equation [40,51,52] r obs H ¼ r diox H � D diox D obs (1) In some cases, we observed that Aβ1-40 peptide samples showed a notable reduction in the overall rate of fibrils formation. We hypothesized that this unexpected behavior could be ascribed to small amount of Aβ1-40 fragments present in the sample and generated by peptide degradation.…”