Consensus DNA Inhibits p53 Aggregation but Fails to Rescue Mutants
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The tumor suppressor p53 plays a crucial role in regulating gene expression under cellular stress. Somatic mutations to its DNA-binding domain are common in lethal cancers and lead to lost regulatory function and pathological hallmarks, such as misfolded p53 aggregates and amyloid-like fibrils. Despite decades of these observations, the molecular determinants driving p53 aggregation and its role in cancer incidence, progression, and lethality remain poorly defined. Identifying these determinants, however, is critical for developing therapeutic interventions that modulate protein solubility in cancer and understanding disparities in cancer outcomes. Therefore, we investigated whether consensus DNA sequences that stabilize wild-type p53 are sufficient to regulate the aggregation of oncogenic p53 mutants with reduced binding affinities. Using a combination of probe-based and label-free spectroscopic and microscopic techniques, we examined how consensus (p21, Bax) and non-consensus (p21-scramble, Poly-GC) DNA oligonucleotides regulate the aggregation of wild-type p53 in comparison to three cancer-associated mutants (R248Q, R273H, and R175H). We find that equimolar p21 consensus sequences significantly inhibits wild-type p53 aggregation, while other DNA sequences do not. In contrast, oncogenic p53 mutants evaded DNA regulation of protein aggregation, and some oligonucleotides even enhanced aggregation at low concentrations, which suggests concentration-dependent DNA-p53 interactions. These findings emphasize that DNA response elements are sufficient for regulating wild-type p53 aggregation in solution. However, key somatic mutations in cancer promote aggregation at the expense of DNA-binding, directly leading to the loss of p53 solubility through biochemical interactions. Taken together, these observations suggest that potent regulators of p53 aggregation should aim to restore affinity between oncogenic p53 mutants and regulatory DNA to minimize the pathological hallmarks of lethal tumors.
Significance Statement
Our research investigates whether DNA sequences that bind tumor suppressor protein p53 regulate its aggregation behavior in wild-type and somatically mutated forms linked to cancer. While specific DNA sequences regulate p53 aggregation, protein mutations that reduce affinity for consensus DNA or destabilize p53’s conformation result in persistent, likely dysfunctional aggregates, even in the presence of regulatory nucleotides. Unsurprisingly, p53 and it’s mutants exhibit increased aggregation when there is substantially more protein to DNA, suggesting insufficient regulatory DNA or excess unregulated p53 may inevitably lead to pathological aggregation. These findings clarify our understanding of p53’s molecular behavior and suggest that cancer-linked somatic mutations enable p53 to evade DNA’s regulatory effects and subsequently aggregate, leading to downstream losses in function.
