Mutational Hotspots and Conserved Domains in P53 Tumour Suppressor Protein

Introduction: The tumour suppressor protein p53 commonly referred to as guardian of the genome plays important role in preserving the genome through the regulation of programmed cell death, DNA repair, energy metabolism, cell cycle entry or exit and senescence. Mutations in p53 can either result to a loss of tumour suppressor function or gain of oncogenic properties. Hence, mutations in p53 are the most frequent genetic mutational alteration in human cancers, associated with worse prognosis and more aggressive disease outcome. Methods: To assess the mutational hotspots and conserved regions of p53, I analyzed 76 complete p53 protein sequences covering whole exons from the NCBI GenBank database. Multiple sequence alignment (MSA) was done using ClustalX version 2.1. Results: Thirty-five (19) mutations were identified with more frequent mutations in amino acid (aa) position 72 and 79 (Exon 4), amino acid deletion in codon 112-122 (Exon 4), codon 213 (Exon 6), codon 248 (Exon 7), codon 273 (Exon 8) and codon 278 (Exon 8). Mutations at amino acid position 79, 248, 278 located in the DNA-binding domain exhibited more than one alteration in same position. Conclusions: Findings from this study revealed the prevalence of mutations in the DNA binding domain of p53 and the structure-function effect of the mutations. Assessing the pattern and frequency of p53 alterations, and analyzing it thoroughly for each carrier, could help in identifying correlations between p53 status and outcome and possible candidate for gene therapy.


Introduction
The p53 gene is a very crucial tumour suppressor gene, commonly referred to as guardian of the genome because of its role in conserving genomic stability. The tumour suppressor, p53 gene, a 53-kDa nuclear phosphoprotein, composed of 19180bp, spans 11 exons and 10 introns which codes for 393 amino acids is located on the short arm of human chromosome 17 (17p13.1 locus), structurally and functionally divided to distinct domains; the transactivation domain (TAD); proline rich domain; DNA-binding domain (DBD); oligomerization domain; and c-terminus regulatory domain (CRD) 1,2 . Exon 1 is non-coding but notable for the very large intron that comes after it and the ability to form a tightly bound stem-loop structure to wild type p53, while exons 2 and 3 encodes the TAD; a serinethreonine-rich region which permits the interaction of p53 with MDM2 (murine double minute 2) and other proteins and a phosphorylation site by ATM. Exons 4 to 8 encodes the DNA binding domain and majority of reported mutations in p53 are in this region 2 . Exons 9 and 10 encode the oligomerization domain, which is responsible for the interaction with Rad51 and BRCA1 and formation of an active tetramer 3 . Functionally, p53 plays important role in programmed cell death (apoptosis), cell cycle regulation, angiogenesis, cellular stress response, cell proliferation, DNA repair by activating GADD 45 (Growth arrest and DNA damage) transcription and binding to ERCC3, modulation of senescence in response to cellular insult and ageing [4][5][6][7] .
In addition to its role in cell cycle regulation, facilitating DNA repair and programmed cell death, p53 also plays important role in the antioxidant and energy metabolism regulation 8,9 . Tumour cells depend largely on energy metabolism as precursors for macromolecule biosynthesis, in order to cater for their rapid growth and proliferation requirement. Reactive oxygen species (ROS) generated through oxidative stress can initiate cellular insult resulting to DNA damage. P53 in a crosstalk controls intracellular ROS by up-regulating nuclear factor erythroid-related factor 2 (NRF2) in response to mild stress and inhibits NRF2 in response to severe stress, in order to induce apoptosis. In mild stress scenario, NRF2 links with p21 in the nucleus and transactivate antioxidant enzymes to stabilize intracellular ROS level 4,8 . Since first published findings on p53 mutations involvement in carcinogenesis in 1989 10 , mutations in p53 have been a subject of interest in cancer biology, being the most frequent genetic mutational alteration accounting for more than 50% of cases in human cancers 6,11,12 . Mutations in p53 have been associated with worse prognosis and more aggressive disease outcome in several cancer types 6,[12][13][14] which are also reported in the International Agency for Research on Cancer (IARC) database 15 . High frequency mutations in p53 resulting to increased risk, chemoresistance and poor prognosis have been reported in endometriosis 16 , skin cancer 17 , breast cancer 18 , cervical cancer 9,19,20 , testicular cancer 21 , colorectal cancer 2,5 and many other cancer types 6,9,11,22 . Earlier studies have identified missense mutation as the most prevalent in p53, targeting exons 5-8 which codes for the DNA binding domain 8,14 .
Most mutations observed in p53 impair its DNA-binding ability, thereby allowing cellular proliferation in state where cells with intact p53 function are regulated or suppressed 6,8 .
Hence, during cellular insult or damage, cells with mutated p53 are unable to initiate cell cycle arrest, DNA repair or apoptosis, thereby resulting to unregulated proliferation, metastasis, and invasion of damaged cells 2,12,23 . Mutation pattern in p53 can help understand the contribution of endogenous events and exogenous agents in carcinogenesis or progression. This may help with respect to diagnosis, detection and in definitive approach to cancer therapy. Thus, this study was carried out using multiple sequence alignment (MSA) to investigate the frequency of mutations, mutational hotspots and conserved domains in p53 spanning exons 1 through 11. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 August 2020 doi:10.20944/preprints202008.0562.v1

Data acquisition
From the available p53 sequence in NCBI database, total 76 p53 protein sequences fit into the inclusive criteria for this study; filtered as "high coverage only, Homo sapiens, complete (393 aa), > 97% percent identity, all exons, and low coverage excl" were retrieved from the NCBI database (https://ncbi.nim.nih.gov/protein/?term꞊P53). The protein sequences in the NCBI database used in this study are listed in Table S1 (with accession numbers with corresponding amino acid sequences in FASTA format).

Multiple sequence alignment (MSA)
The retrieved p53 protein sequences were aligned with the reference protein 24 by ClustalX version 2.1 25 MSA software using the default parameters 26 .

Sequence and mutational analysis
In this study we used the filtered data to analyze the polymorphisms and mutations in the 76 p53 protein sequences in order to identify similar or conserved domains and mutational patterns. Mutations that occur multiple times independently were focused on as they are likely candidates for understanding the mutational landscape in p53 while 28 mutation sites with only a single mutation candidate were not fully explored. After exclusion, we observed mutations in multiple sequences at 7 different positions spread across exons 4, 6, 7, and 8 in the p53 protein sequence.

Results
The result of the MSA of the protein sequences with NP_001119584.1 as reference protein sequence using ClustalX version 2.1. Mutations observed are reported in Table 1     sensitivity to HPV-E6-mediated ubiquitin-dependent proteolysis 15,29 . The allelic frequencies, 0.54p53Arg and 0.46p53Pro, found in this study, corresponded with those described in earlier reports 1,9,16,30

Conclusion
The present study revealed the hotspot mutations in p53 protein sequences and their impact to the structure-function as the guardian of the genome. The results showed the prevalence of missense mutations as the most recurrent mutation type in p53. Synonymous (silent) mutations do not change the amino acid, hence, this study could not account for them due to

Declaration of Competing Interest
The author declare no conflict of interest