Preface

Genetic information within the cell is contained and stably inherited in the form of deoxyribonucleic acid (DNA). To ensure faithful transmission of this genetic information, it is important for the cell to accurately copy this information. This is followed by the division of old cells into daughter cells. Both processes are essential to maintain the integrity and functionality of the cells. Also, properly controlled cell division is essential to maintain cells in a healthy state, and perturbances in this process lead to the transformation of healthy cells into malignant ones. One of the ways through which the formation of new cells is properly controlled is regulation with the help of specialized proteins, such as p53. This protein plays its cell protective role under the most widely studied conditions and this characteristic lends it its name of "the guardian of the genome." Additionally, p53 is also involved in an array of other functions. With advancements in our knowledge due to the development of new scientific techniques, we have come to appreciate many more roles of this protein, ranging from the prevention of cancer to its role as an environmental biomarker.

This book highlights p53's vast array of functions in a cell, including its lesser-known roles. It is divided into three sections. Section 1 includes an introductory chapter (Chapter 1) on p53. Section 2 includes chapters on the role of p53 in human cancers (Chapter 2), in DNA repair (Chapter 3), and in gene regulation and gene therapy (Chapter 4). Section 3 includes a chapter on the role of p53 as an environmental biomarker (Chapter 5) and a chapter on the study of p53 at a single molecule level (Chapter 6), revealing the dynamics and energetics of p53 binding to DNA.

This book answers some of the most fundamental as well as some of the most obscure questions about p53. We hope it elicits interest in research to uncover and shed light on other uncharacterized functions of this protein.

> **Mumtaz Anwar** Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago, Chicago, USA

> > **Zeenat Farooq**

Postdoctoral Research Associate, Division of Endocrinology and Metabolism, Department of Medicine, University of Illinois at Chicago, Chicago, USA

#### **Mohammad Tauseef, MPharm Ph.D.**

Associate Professor, Department of Pharmaceutical Sciences, Chicago State University, Chicago, USA

#### **Vijay Avin Balaji Ragunathrao, Ph.D.**

Assistant Professor (Research), Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago, Chicago, USA

Section 1 Introduction

#### **Chapter 1**

## Introductory Chapter: p53 - The Miracle Protein That Holds the Distinction of Being "Guardian of the Genome"

*Zeenat Farooq and Mumtaz Anwar*

### **1. Introduction**

P53 is a protein encoded by TP53 gene in humans. This gene is located on the short arm of chromosome 17 in humans [1]. The gene contains 11 exons and several regulatory regions. The gene is highly conversed in nature and is found across invertebrate and vertebrate species. However, there is a high degree of variability in the coding sequence of p53 in vertebrate and invertebrates. The protein encoded by TP53 is typically known as p53 because in earlier days (around 1979), it appeared to localize at around 53 KDa on a sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. However, it was later found that the protein is smaller in size and the lag in migration in the gel occurred due to the abundance of proline residues that cause a *kink* in the structure. The actual mass of the protein, based on summation of molecular masses of all the amino acid contained is around 43.7KDa [2]. Many terms are used for the identification of p53-like tumor protein p53, tumor suppressor p53, phosphoprotein p53, and so on. By far, the most significant working definition offered by any term for its identification is *p53, the guardian of the genome*. This term inherits its "*guardian status*" by the fact that p53 plays a crucial and quintessential role in guarding (protecting) the genome against damage and is therefore found to be mutated in many forms of cancer. In fact, it holds the title of being the most frequently mutated gene in all cancers, documented to be mutated in more than 50% of all cancers [3]. The protein performs its guardian role by acting as a transcription factor and regulating the expression of various genes.

In its three-dimensional structure, p53 protein consists of the following domains, briefly described from N to C terminus below (**Figure 1**) [4–6].


In its ground state, p53 exists inside the cells in the form of a complex with another protein mdm2 (HDM2 in humans). This dimeric association holds p53 in an inactive state. Mdm2 is also a ubiquitin ligase, which ubiquitylates p53 and marks it for proteolytic degradation. In this manner, p53 undergoes a continuous turn-over in the cells, with a half-life of about 20 minutes. Upon activation, p53 dissociates from mdm2 and becomes available to contribute to a myriad of cellular functions. It exists as a tetramer in its active state. The most common mechanism of p53 activation is phosphorylation at multiple residues.

Upon activation of a stress-signaling cascade in a cell-like DNA damage, activation of proto-oncogenes, or apoptotic pathways, p53 becomes phosphorylated by a variety of kinases, each activated by a particular type of stress signal. Phosphorylation of p53 brings about a conformational change in the protein that interferes with its binding to mdm2 and instead promotes oligomerization of p53. Afterward, p53 moves into the nucleus with the help of NLS and binds to its target genes to promote their transcription. The kinase enzymes therefore favor p53 function in two ways (**Figure 2**).

**Figure 1.**

*Domain structure of p53 showing its various domains and their relative size from N to C terminal.*

#### **Figure 2.**

*Outline model depicting the effect of p53 on cells upon activation, leading to cell cycle arrest and DNA repair. If the repair fails, p53 activates pro-apoptotic genes to embark the cells on the path of apoptosis.*

*Introductory Chapter: p53 - The Miracle Protein That Holds the Distinction of Being "Guardian… DOI: http://dx.doi.org/10.5772/intechopen.101918*


p53 kinases fall into two major groups. Additionally, oncogenes can also activate p53.


#### **2. Cellular roles of p53**

The quintessential roles of p53 within the cells are as follows [7].

#### **2.1 DNA damage and repair**

Upon sensing DNA damage as a result of genotoxic insults, kinases such as ATM and ATR become activated and phosphorylate p53. p53, in turn, activates transcription of proteins that lead to cell cycle arrest at G1/S phase. This allows enough time for the DNA repair proteins to repair the damaged DNA. This process ensures that damaged DNA does not replicate and become inherited by daughter cells through cell division. Once the repair is complete, the cell goes back to the unstimulated state and starts diving normally.

#### **2.2 Apoptosis**

The term apoptosis refers to programmed cell death. It is a process by which damaged cells undergo a carefully orchestrated signaling program that culminates in death of the cells without harming neighboring healthy cells of the tissue. This phenomenon occurs when a cell accumulates damage to such an extent that repair is not possible. p53 plays a very critical role in initiating apoptosis of such cells. Both processes are interrelated.

Because of the central role played by p53 in maintaining cellular homeostasis and genome integrity, mutations in the gene are detrimental for p53 function. A large number of mutations have been identified in the gene, which result in the formation of a mutant p53 protein that no longer retains its DNA-binding or oligomerization ability, leading to loss of function. Some mutations have been observed in the DNAbinding domain, which affect binding of p53 to its target genes. Other mutations in the oligomerization prevent p53 sub-units from coming together and forming a functional, oligomeric transcription factor. Another aspect of such mutations is that a single-mutant p53 subunit can prevent oligomerization of wild-type subunits, exerting a dominant negative effect. All these mutations have been identified in many forms of cancer. Additionally, p53 promoter has been shown to undergo an increase in promoter methylation, which leads to decrease in its expression. This

mechanism of epigenetic regulation of p53 expression was first of all demonstrated by Bird et al. and has ever since been observed in various other forms of cancer [8–10]. The phenomenon of increase in promoter DNA methylation to decrease expression of cognate gene is also identified as a key epigenetic mechanism with a wide array of cellular functions [11]. According to some reports, it is not only the dissociation of p53 from mdm2 which increases its half-life and cellular availability but some signaling cascades stimulate the translation of p53 mRNAs to increase cellular levels. Increase in mRNA translation of p53 has also been observed to take place in stem cells to trigger differentiation [12]. With the availability of better techniques to carry out research, more exciting work on p53 is being carried out and published, which sheds light on newer and exciting functions of p53.

This book focuses on the roles of p53 as a guardian of genome, explaining in detail various roles performed by the protein under different physiological conditions. The following chapters talk at length about different facets of p53, each related to its cell protective function in light of both established phenomena and latest research in the field on p53.

### **Author details**

Zeenat Farooq and Mumtaz Anwar\* Department of Pharmacology and Regenerative Medicine, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

\*Address all correspondence to: mumtazan@uic.edu; mumtaz\_anwar1985@yahoo.co.in

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: p53 - The Miracle Protein That Holds the Distinction of Being "Guardian… DOI: http://dx.doi.org/10.5772/intechopen.101918*

#### **References**

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[2] Ziemer MA, Mason A, Carlson DM. Cell-free translations of proline-rich protein mRNAs. The Journal of Biological Chemistry. 1982;**257**(18): 11176-11180

[3] Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sørlie T, et al. Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Research. 1994;**22**(17):3551-3555

[4] Venot C, Maratrat M, Dureuil C, Conseiller E, Bracco L, Debussche L. The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression. The EMBO Journal. 1998;**17**(16):4668-4679

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[7] Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;**88**(3):323-331

[8] Malhotra P, Anwar M, Nanda N, Kochhar R, Wig JD, Vaiphei K, et al. Alterations in K-ras, APC and p53 multiple genetic pathway in colorectal cancer among Indians. Tumour Biology. 2013;**34**(3):1901-1911

[9] Bird AP. CpG-rich islands and the function of DNA methylation. Nature. 1986;**321**(6067):209-213

[10] Farooq Z, Shah A, Tauseef M, Rather RA, Anwar M. Evolution of Epigenome as the Blueprint for Carcinogenesis. Rijeka: IntechOpen; 2021. DOI: 10.5772/intechopen.97379

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### Section 2
