The p53 tumor suppressor is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, DNA repair, cellular senescence, metabolism and tumor suppression. It is maintained at low levels in unstressed cells, but becomes stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 functions. Previously, we characterized the complexes formed between the Taz2 domain of the transcriptional co-activator p300 and either the first (TAD1, residues 1-40) or second (TAD2, residues 35-59) transactivation subdomains of p53. Our results showed that both TAD1 and TAD2 occupy the same region of Taz2, form short alpha helices when bound, have similar affinities for Taz2, and are stabilized by both hydrophobic and electrostatic interactions. Although both TAD1 and TAD2 bind to various domains within p300, they also interact with distinct proteins and can function independently of one another. These results suggest the existence of distinguishing transcriptional cofactors for TAD1 and TAD2 whose interaction is regulated differently by p53 phosphorylation. Comparison of the structures of the two complexes also suggests that these two similar domains within p53 may function differently in co-activator recruitment after stress. Therefore, we have initiated a project to identify differential interactors of TAD1 and TAD2 in response to cellular stress that are modulated by p53 phosphorylation. We have synthesized a series of p53 peptides representing either TAD1 or TAD2 to use as bait for pulldown of interacting proteins from nuclear extracts prepared from cells treated to cellular stress; mass spectrometry analysis will be used to identify and quantitatively compare the interactors to discriminate between those that preferentially interact with TAD1 and TAD2. Our preliminary experiments have identified a list of potential interactors that are uniquely pulled down by TAD1 when it is phosphorylated on Thr18. Investigations into those proteins identified several that had previously been shown to interact with p53. Among those that had not been shown to interact with p53 were proteins involved in DNA repair and DNA damage checkpoint signaling. The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, neddylation and hydroxylation that are primarily localized to the terminal thirty residues of the protein. We are interested in understanding the specific effects of individual site-specific modifications and the interplay between them. We have reported that p53 can be both mono- and dimethylated on Lys382, with the former modification repressing p53 transcriptional activity and the latter promoting DNA repair. It was recently reported by Wang et al. (PLoS Biol, 2014, 12:e1001819) that p53 Lys382 can be hydroxylated by JMJD6 to negatively regulate p53 activity. We have previously shown that this site can also be mono- and di-methylated, in addition to demonstrated acetylation and ubiquitination of Lys382. We are currently investigating the differential effects of these modifications on p53 conformation and interactions. We are synthesizing long synthetic peptides containing specific PTMs on Lys382 that will be used in biochemical and biophysical assays. A number of p53 point mutations have been reported to occur in approximately half of all tumors. These mutations leave p53 unable to function as a transcription factor and prevent tumor growth. Moreover, many mutant forms of p53 have novel oncogenic activities due to a gain-of-function mechanism. Therefore, mutant p53 is an important target for the development of an agent to improve the response to anti-cancer treatments. Although a number of screening trials have identified small organic molecules that are able to bind to mutant p53 in order to stabilize its native fold and revert its inactivation, these screens have been large-scale efforts that did not made use of existent structural knowledge of mutant p53 to identify a molecule that could act selectively on entire groups of p53 mutations. One characteristic of several p53 mutants is their structural instability with partial unfolding and the formation of aggregates similar to those seen in amyloid diseases, thus resulting in protein inactivation. Recently, Eisenberg and colleagues (Soragni et al., Cancer Cell 2016, 29, 90-103) used a peptide with a single point mutation derived from the sequence of p53 residues 252-258 to rescue p53 function and reduce tumor growth in mouse models of ovarian carcinoma. This approach is based on the ability of the peptide to mask the native protein sequence that nucleates aggregation. As the highly aggregation-prone and hydrophobic nature of the sequence limits its use as a p53 aggregation inhibitor alone, there is a further need for the development of improved molecules with more favorable properties. We have initiated a study to evaluate the potential of peptide mimetics alone and in combination with nutlin-3a, an inhibitor of the interaction of p53 with its negative regulator MDM2, to specifically reactivate mutant p53 and induce cell cycle arrest and apoptosis through the wild-type p53 pathway. We have designed and synthesized several new molecules, and our preliminary data demonstrates that the lead peptide mimetic inhibits the formation of amyloid-like fibrils by a segment of p53 (residues 252-258) in vitro. As this segment is highly conserved in the DNA-binding domain of p53, p63 and p73, it offers a potential robust target for inhibition of the aggregation of mutant p53 and a novel means to treat tumors with mutant p53.
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