Propagation of aggregated p53: Cross-reaction and coaggregation vs. seeding

Significance Aggregation of tumor suppressor p53 oncogenic mutants not only loses their activity but may also lead to gain of oncogenic function, possibly by coaggregation with other proteins. We explored how a destabilized p53 mutant may coaggregate with WT p53 and its homologs p63 and p73. The results are explained by the simple two-step initiation mechanism we proposed. Rather than the oncogenic mutant seeding coaggregation by a prion-like process, coaggregation results from the simultaneous unfolding and cross-reaction of WT and mutant molecules. Consequently, preformed p53 aggregate displays little seeding of aggregation of p53, and high concentrations of p53 mutants are required to trap WT p53 into aggregate. Coaggregation is predominantly by trapping rather than seeding and induced propagation. Destabilized mutant p53s coaggregate with WT p53, p63, and p73 in cancer cell lines. We found that stoichiometric amounts of aggregation-prone mutants induced only small amounts of WT p53 to coaggregate, and preformed aggregates did not significantly seed the aggregation of bulk protein. Similarly, p53 mutants trapped only small amounts of p63 and p73 into their p53 aggregates. Tetrameric full-length protein aggregated at similar rates and kinetics to isolated core domains, but there was some induced aggregation of WT by mutants in hetero-tetramers. p53 aggregation thus differs from the usual formation of amyloid fibril or prion aggregates where tiny amounts of preformed aggregate rapidly seed further aggregation. The proposed aggregation mechanism of p53 of rate-determining sequential unfolding and combination of two molecules accounts for the difference. A molecule of fast-unfolding mutant preferentially reacts with another molecule of mutant and only occasionally traps a slower unfolding WT molecule. The mutant population rapidly self-aggregates before much WT protein is depleted. Subsequently, WT protein self-aggregates at its normal rate. However, the continual production of mutant p53 in a cancer cell would gradually trap more and more WT and other proteins, accounting for the observations of coaggregates in vivo. The mechanism corresponds more to trapping by cross-reaction and coaggregation rather than classical seeding and growth.