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. Earlier, we characterized the complex formed between the first subdomain of the N-terminal transactivation domain of p53 (TAD1, residues 1-40) and the Taz2 domain of the transcriptional co-activator p300 and also showed that the second subdomain, TAD2, competes with TAD1 for binding to Taz2. Recently, we solved the NMR solution structure of Taz2 in complex with the second transactivation subdomain of p53 (residues 35-59). 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. However, the orientations of the two alpha helices differ. Primary among the hydrophobic interactions of TAD2 and Taz2 is the packing of the p53 Ile50 side chain in a deep hydrophobic pocket in the core of Taz2. Interestingly, p53 amino acids (35-59) contain seven acidic residues that form a series of salt bridges and hydrogen bonds with formal and partial positively-charged residues of Taz2. Ser46 and Thr55, which are subject to post-translational phosphorylation and bracket the ends of the bound helix, are mostly unbound, consistent with our previous observation that phosphorylation does not significantly affect binding. Comparison of the structures of the two complexes suggests that these two similar domains within p53 may function differently in co-activator recruitment after stress. In addition, it provides new understanding of the importance of flexibility in this domain for the formation of critical protein-protein interactions. The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, and neddylation, that are primarily localized to the terminal thirty residues of the protein. We have been 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. p53 dimethylated on Lys382 is preferentially bound by the Tandem Tudor domain (TTD) of 53BP1, promoting DNA repair. Recently, we found that acetylation of Lys381 and dimethylation of Lys382 occurs during the response to DNA damage and alters the binding of p53 to 53BP1. This dual PTM causes a significant conformational change in the bound form of p53, converting the U-shaped p53K382me2 peptide into an alpha-helical conformation for the p53K381acK382me2 peptide. Other PTMs, including phosphorylation of serine and threonine residues of p53, further influence the interaction with the TTD of 53BP1. Our data suggest a novel p53 regulatory mechanism in which different combinations of PTHs enable distinct conformations in p53 when bound to specific interactors. The acetylation-methylation interplay can function as a switch, allowing for distinctly different p53 responses to severe DNA damage as opposed to transient low-level DNA breaks that occur during normal cell processes. Recent studies have shown that the association of 53BP1 TTD with the methylated histone PTMs is necessary but not sufficient for rapidly recruiting 53BP1 to damaged DNA; p53 recruitment further requires self-association of 53BP1 through its oligomerization domain, located upstream of the TTD. The juxtaposition of multiple TTDs through 53BP1 oligomerization raises the possibility of mechanistic and functional crosstalk between the dimethyllysine PTMs. We have investigated the recognition by 53BP1 TTD of two distinct dimethyl lysine-containing sequences, p53K370me2 and p53K382me2. Analysis of binding affinities and x-ray structures of cross-linked TTDs suggests that oligomerized 53BP1 can recognize simultaneously the dual K370me2/K382me2 sequences on a p53 substrate, with one monomer binding to each modified lysine. A similar complex could form in which one monomer binds dimethylated p53 and a second monomer binds dimethylated histone tails, providing a mechanism for the localization of p53 near sites of DSB repair. One of the naturally expressed isoforms of p53, deltaNp53, lacks the first transactivation domain (TAD1) of p53 but does contain the second transactivation domain (TAD2). The expression and stability of the two proteins are affected differently by cell type, cell cycle phase and exposure to various stresses. p53 and deltaNp53 form heterotetramers and the relative abundance of deltaNp53 influences the transactivation activity and target gene specificity of p53. Another naturally occurring isoform, delta133p53, lacks both TAD1 and TAD2 and negatively regulates the activity of full-length p53. We found that during replicative senescence, ubiquitination of both lysine 381 and lysine 382 by the chaperone-associated E3 ubiquitin ligase STUB1 leads to autophagic degradation of delta133p53, thus identifying a p53 isoform-specific protein turnover mechanism. Therefore, delta133p53 represents a functional and regulatory link between cellular senescence and autophagy, two cellular phenotypes involved in aging and cancer. Global effects of p53 PTM mouse models containing missense mutations at p53 PTM sites have been used to investigate the complex effects of p53 PTMs in a physiological setting. Our quantitative mass spectrometry studies of Ser18Ala knock-in mice demonstrated that the mutation affected proteins with roles in energy and metabolism pathways following ionizing radiation. As p53 has been shown to have important roles in regulation of metabolism and energy pathways, we have initiated studies to further understand the modulation of p53-dependent effects on metabolism. As a complement to mouse models, we are using genomic editing techniques for introducing specific modifications into genes in human cells. These methods will allow a more comprehensive investigation of the interrelationships between p53 PTMs and metabolism.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Cooks, Tomer; Pateras, Ioannis S; Jenkins, Lisa M et al. (2018) Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246. Nat Commun 9:771
Veschi, Veronica; Liu, Zhihui; Voss, Ty C et al. (2017) Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma. Cancer Cell 31:50-63
Mazur, Sharlyn J; Gallagher, Elyssia S; Debnath, Subrata et al. (2017) Conformational Changes in Active and Inactive States of Human PP2C? Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry. Biochemistry 56:2676-2689
Zhang, Zhan; Liu, Ling; Gomez-Casal, Roberto et al. (2016) Targeting cancer stem cells with p53 modulators. Oncotarget 7:45079-45093
Tong, Qiong; Mazur, Sharlyn J; Rincon-Arano, Hector et al. (2015) An acetyl-methyl switch drives a conformational change in p53. Structure 23:322-31
Tong, Qiong; Cui, Gaofeng; Botuyan, Maria Victoria et al. (2015) Structural plasticity of methyllysine recognition by the tandem tudor domain of 53BP1. Structure 23:312-21
Lu, X; Mazur, S J; Lin, T et al. (2014) The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis. Oncogene 33:2655-64
Jenkins, Lisa M Miller; Durell, Stewart R; Mazur, Sharlyn J et al. (2012) p53 N-terminal phosphorylation: a defining layer of complex regulation. Carcinogenesis 33:1441-9
Karamychev, Valeri N; Wang, Difei; Mazur, Sharlyn J et al. (2012) Radioprobing the conformation of DNA in a p53-DNA complex. Int J Radiat Biol 88:1039-45
Fujita, Kaori; Horikawa, Izumi; Mondal, Abdul M et al. (2010) Positive feedback between p53 and TRF2 during telomere-damage signalling and cellular senescence. Nat Cell Biol 12:1205-12

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