Multisite aggregation of p53 and implications for drug rescue

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Protein aggregation is implicated in various diseases, often forming regular fibrils from specific sequences.
• Oncogenic mutants of tumor suppressor p53 aggregate into amorphous fibrils, with a peptide near Ile254 proposed as a key aggregation driver.
• Understanding the mechanisms of amorphous protein aggregation is crucial for disease intervention.

Purpose of the Study:

• To investigate the aggregation mechanisms of mutant p53 and identify aggregation-driving sequences.
• To evaluate the efficacy of inhibiting specific aggregation sites in vitro and in vivo.
• To explore the therapeutic potential of peptides targeting aggregation-driving sequences in cancer cells.

Main Methods:

• Limited proteolysis of harvested aggregates to identify aggregation sites.
• Site-directed mutagenesis to assess the impact of mutations on aggregation kinetics and products.
• In vitro aggregation assays and cell-based cytotoxicity studies using engineered peptides.

Main Results:

• Multiple aggregation-prone sites were identified, and amorphous aggregate formation was linked to multisite branching after slow protein unfolding.
• Mutating individual aggregation sites, including the Ile254-surrounding peptide, did not prevent in vitro aggregation due to compensatory aggregation via other sites.
• Peptides targeting identified aggregation sequences, including Ile254, effectively killed cancer cells, independent of p53 knockdown, indicating potential non-p53 targets or altered protein interactions.

Conclusions:

• Amorphous aggregation of large proteins like mutant p53 may result from multisite branching after unfolding.
• Inhibiting single aggregation sites may be insufficient to prevent aggregation in vivo.
• Targeted peptides show promise for cancer therapy by inducing rapid cell death through mechanisms potentially independent of p53's transcriptional activity.