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

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, 5, 6].

• N-terminus transcription activation domain (TAD) or activation domain 1 (AD1). It is rich in acidic residues. It plays role in regulation of pro-apoptotic genes.

• Activation domain 2 (AD2). It is important for apoptotic activity of p53.

• Proline-rich domain. It is responsible for lag in migration on SDS-PAGE.

• DNA-binding domain (DBD). It plays role in binding to DNA elements on target genes.

• Nuclear localization signal (NLS). It consists of a group of amino acids that are involved in localization of the protein into the nucleus through nuclear pore.

• Self-oligomerization domain (OD). Oligomerization is important for self-annealing and activity of p53.

• C-terminal domain that antagonizes the function of DNA-binding domain.

Figure 1.

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

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).

• Increase the half-life of p53 by promoting dissociation from mdm2. This increases the cellular concentration of p53 to make it available for the challenge in hand.

• Phosphorylation event favors self-oligomerization, which is essential for p53 activity.

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.

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

• Kinases belonging to MAPK family. These include p38 MAPK, JNK1-3,ERK1-2. These are activated in response to stresses such as membrane damage, oxidative stress, heat shock, osmotic shock, etc.

• Kinases belonging to ATM family. These include ATM, ATR, CHK1, CHK2, DNA-PK. These are activated as a result of cell cycle checkpoint responses induced due to DNA damage.

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 DNA-binding 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, 9, 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.