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Multisite aggregation of p53 and implications for drug rescue.

ABSTRACT: Protein aggregation is involved in many diseases. Often, a unique aggregation-prone sequence polymerizes to form regular fibrils. Many oncogenic mutants of the tumor suppressor p53 rapidly aggregate but form amorphous fibrils. A peptide surrounding Ile254 is proposed to be the aggregation-driving sequence in cells. We identified several different aggregating sites from limited proteolysis of harvested aggregates and effects of mutations on kinetics and products of aggregation. We present a model whereby the amorphous nature of the aggregates results from multisite branching of polymerization after slow unfolding of the protein, which may be a common feature of aggregation of large proteins. Greatly lowering the aggregation propensity of any one single site, including the site of Ile254, by mutation did not inhibit aggregation in vitro because aggregation could still occur via the other sites. Inhibition of an individual site is, accordingly, potentially unable to prevent aggregation in vivo. However, cancer cells are specifically killed by peptides designed to inhibit the Ile254 sequence and further aggregation-driving sequences that we have found. Consistent with our proposed mechanism of aggregation, we found that such peptides did not inhibit aggregation of mutant p53 in vitro. The cytotoxicity was not eliminated by knockdown of p53 in 2D cancer cell cultures. The peptides caused rapid cell death, much faster than usually expected for p53-mediated transcription-dependent apoptosis. There may also be non-p53 targets for those peptides in cancer cells, such as p63, or the peptides may alter other interactions of partly denatured p53 with receptors.

SUBMITTER: Wang G

PROVIDER: S-EPMC5380089 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Multisite aggregation of p53 and implications for drug rescue.

Wang GuoZhen G Fersht Alan R AR

Proceedings of the National Academy of Sciences of the United States of America 20170314 13

Protein aggregation is involved in many diseases. Often, a unique aggregation-prone sequence polymerizes to form regular fibrils. Many oncogenic mutants of the tumor suppressor p53 rapidly aggregate but form amorphous fibrils. A peptide surrounding Ile254 is proposed to be the aggregation-driving sequence in cells. We identified several different aggregating sites from limited proteolysis of harvested aggregates and effects of mutations on kinetics and products of aggregation. We present a model ...[more]

PMID: 28292898

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Project description:To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6?kcal?mol(-1) (15.1?kJ?mol(-1)). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the ?-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.

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Multisite aggregation of p53 and implications for drug rescue.

Publications

Multisite aggregation of p53 and implications for drug rescue.

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