Posttranslational modification and evolution of tetramerization domain in tumor suppressor protein p53

Tumor suppressor protein p53 induces apoptosis and cell cycle arrest in cells that are stimulated by a variety of cellular stresses including genotoxic stress. p53 maintains genomic integrity, and its functions are the most important for resistance to cellular tumorigenesis. Mutations of TP53, which is the p53-encoding gene, are frequently found in malignant tumors. Because apoptosis is not induced in cancer cells with mutations or deletion in TP53 in response to radiation therapy or treatment with anticancer agents, the prognosis of these patients is very poor. In response to cellular stress p53 is stabilized, tetramerized, and activated to exert functions such as transcription activation. This function is precisely regulated by posttranslational modifications such as phosphorylation, acetylation, ubiquitination, and methylation of more than 50 residues. Homotetramerization of the p53 protein through the tetramerization domain (TD) at the C-terminal region is indispensable for its function, and its transcriptional activity is regulated by the stability of the tetrameric structure. The TD of human p53 consists of a β-strand, turn, and α-helix and is in equilibrium between the monomer and tetramer. The regulation by posttranslational modifications in the tetramerization domain is still unclear; thus, detailed elucidation of the regulatory mechanism through posttranslational modifications is required. p53 is found in a wide range of organisms from Lamprey, which is an early vertebrate, to mammals. Though the role of p53 is important in the evolution of vertebrates, the function of p53 and the evolution process of the tetrameric structure are unclear. As well as phylogenetic analysis of the sequence, a comprehensive analysis of the structure and the function is required.  The aim of this study was to understand how the stability of the tetrameric structure is changed by posttranslational modifications and how the TD regulates p53’s activity and function. To this end, I explored the Arg methylation regulatory mechanism and the evolution of the p53 tetramerization domain in vertebrates.   This thesis consists of five chapters. The background of the study and the study goals are described as a general introduction in Chapter 1. I outlined the tumor suppressor function of p53, its tetrameric structure, and its evolution in vertebrates.   In Chapter 2, I described the function control by methylation of the p53 TD. p53’s function is regulated by posttranslational modifications, including the methylation of three Arg residues in the TD. It has been reported that this methylation by protein Arg methyltransferase 5 (PRMT5) promotes cell cycle arrest, but represses apoptosis. To clarify the regulatory mechanism through methylation, I carried out structure stability analysis of the methylated p53 fragment and an in vitro methylation assay with PRMT5. The methylation of the Arg residues destabilized the oligomeric structure, in particular, Arg337 had a large contribution to the destabilization. I identified PRMT5 methylation sites using nLC-MS/MS and revealed a methylation cascade that started with mono-methylation at Arg335. These findings suggest a novel regulatory mechanism via structural stability modulation of the p53 TD. The affinity between the response element of the target gene and the p53 protein appeared to be high with pro-cell cycle arrest genes and low with pro-apoptotic genes. When p53 binds to a response element and controls transcription, the DNA bends. It is thought that even methylated and destabilized p53 can endure bending of pro-cell cycle arrest genes because the affinity is high, but cannot bear bending of the pro-apoptotic genes.   In Chapter 3, I described the evolution of the structural stability of the p53 TD in mammals. The sequence of the mammalian p53 TD varies in 3–10 residues within species. I synthesized the 35-residue fragment of the p53 TD of human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum and analyzed the thermostability of the oligomeric structure using CD spectrometry. The tree shrew resembles a squirrel but classified independent order; there were substitutions of only four residues in comparison with the human variant, and its oligomeric structure has been shown to be more stable than the human one. By analyzing the mutants, it was determined that the substitution causing the stabilization was of Met354 (Gln in the human version). The homology modeling of the tetrameric structure suggests that the Met side chain located in the C-terminal α-helix stabilized the helix structure of the end through the Met side chain of other protein chain via new hydrophobic interactions. Recently, the tree shrew has attracted a lot of attention, as wild tree shrews drink routinely and are susceptible to hepatitis B infection, which has been thought to infect only chimpanzees and humans. Thus, it is now used as a primate-like model. Aldehyde causes DNA damage, and it has been suggested that the tree shrew can maintain genomic integrity because of its stable p53.  In Chapter 4, I described more extensive analysis of the evolution of the structure, stability, and function of the p53 TD in vertebrates. As well as phylogenic tree analysis, this study analyzed structure, stability, and function. The phylogenic analysis showed that the p53 TD of vertebrates had a longer evolution distance than the TDs of p63 and p73, which are p53 family members that formed a tetramer in the same way. In addition, it was revealed that the p53 TD is more diversified than the DNA-binding domain (DBD). I chose 19 species including jawless fish, cartilaginous fish, ray-finned fish, lobe-finned fish, Amphibia, Reptilia, birds, and Mammalia. Though there were substitutions of many residues including in Lamprey, the TD fragment formed a tetramer in all species. I carried out structure stability analysis and transcriptional activity analysis of chimeric p53. Lamprey had low structural stability and activity, and fish such as Zebrafish had high activity due to a stabilization effect of the second helix in the C-terminal region. However, the Coelacanth species reaching Human aiming at an advance to the land gained stability that was high only in the first helix. Because a mutation in the DBD directly affects binding with DNA and the evolution of the TD regulates the activity of p53 indirectly, it is thought that the stability of the tetrameric structure evolved more than the DBD.   In Chapter 5, I described the all-inclusive conclusion of this study. To understand the regulatory mechanism in vivo, it is very important to clarify the stability modulation mechanism of the p53 tetrameric structure. In this study, it was determined that structural stability is regulated by the methylation of the p53 TD. Furthermore, I propose that the target gene choice control model uses the differences in the affinity with the response element and adjustment of the tetrameric structure stability. Furthermore, as well as phylogenetic analysis of the protein, which is expressed in the whole vertebrate, I performed structural stability and functional analysis for the first time. The results suggest that vertebrates adapted to the environment via the stability of the p53 TD, and p53 played an extremely important role in the evolution process.

Keyphrases: p53, Evolution, Tetramerization, Vertebrates, Structure, Stability, PRMT5, Methylation