Mechanism of Action p53

Abstract

P53 is a 393 residue protein in humans made up of five proposed domains, with which the central DNA binding domain with 100-300 sequences very important for the direct binding of p53 in the promoters of its target genes to specific response elements. P53 is a tumor suppressor gene with cellular stress like oxygen deficiency, oxidative stress, radiation and carcinogens substances, is stimulated has major roles in translational regulation and feedback processes. A wide variety of damage signals that relate to the stability, post-translational alteration and recruitment of p53 to binding sites in chromatin which activate the p53 pathway. As a transcriptional activation, p53 mediates transcriptional changes which facilitate cell death, senescence or reversing and protective arrest of the cell cycle. P53 is a protein under intense investigation because it is necessary to prevent tumor, in human tumors have been found to deregulation of p53 activity. On this article study focuses the mechanism of suppressive p53 effects in the response to any stress and correlation of the mutation p53 with different tumor.

Introduction

Firstly, protein 53 is identified by the discovery of the antibodies against SV40 large T antigen from animal carrying tumors produce by SV40-transformed cells is immunoprecipitated an apparent with mass about 53 kDa is identified as p53 [1]. Initially, gene encoding p53 showed low oncogenic activity as a result of overexpression of the p53 protein in mice tumor cells [2]. In 1984, after the first cloning of p53 gene was reported that a finding that fitted with the hypothesis that p53 was a cellular oncogene that could be activated by mutation [1]. Following that, a number of important findings were soon discovered, including p53 may be inactivated by tumor Viruses. In 1989, p53 was established as tumor suppressor gene after review verified the capacity of wild type 53 to suppress E1A and Ras mediated transformation. Until 1989, it was classified as a tumor suppressor [3]. The first set of such studies on mice expressing temporally regulated version has been proved inhibition tumor genesis of p53 when the exposure to acute DNA damage [4].

1- Structure of P53

P53 gene is located on chromosome 17 is composed of 11 exons of the human genome which coding the p53 protein. The natural p53 protein transcribed and translated consists of 393 amino acids with half-life between 6 to 20 min [5]. The p53 is consisted of four major functional regions, as tetramer-like structure. The N-terminal region consists of the transactivation field (TAD). TAD is the fundamentally n-terminal area. TF11D acts in this location through connecting to TATA box in the promoter and initiate the transcription. TAD divided into TAD1 and TAD2.The follow part is the proline-rich with five PXXP motifs. Then the core domain which highly specialized region responsible for binding DNA sequence, where the inactivating mutations are most commonly identified. Lastly, there is a C-terminal domain, composed of NLS and NES, binding with nonspecific nucleic acid or other protein, target for regulation p53 and posttranslational alteration. The terminal tetramerization domain (TD) is situated between the C-terminal region and DNA-binding domain [6]. DNA binding domain is necessary for specific binding of p53 to its target promoters. In many human cancers the missense mutations within this central domain commonly happen [7]. The mostly mutations occur in disturbing of the p53 capacity to DNA binding or alteration in domain folding. Therefore, these mutation p53 prevents to perform its transcription factor activity [8].

2- Functions of p53 protein in the Cell Bodies

The p53 tumor suppressor is highly susceptible for various genotoxic stress such as ionizing radiation, UV radiation, application of cytotoxic drugs or chemotherapeutic agents, and infectious virus), heat shock, hypoxia, and oncogene overexpression and damage to DNA. Distinct signaling processes have evolved which in effect up regulate different pathways to increase health and survival of organism [9,10]. P53 can activate a variety of responses, including cell cycle arrest, altered metabolism, and restoration of DNA, antioxidant, anti-aging, senescence and apoptosis effects [11].

2-1- Mechanism of the Cell Cycle Arrest

Cell Cycle

The cell cycle is divided into two phases; interphase, which consists from G1, S, and G2 phases, and the mitotic

(M) phase. The end of cytokinesis in a previous division is named as G1 phase which the cell grows in preparation for DNA replication. In S- or synthetic-stage is replicated of chromosomal DNA and two strands of DNA produce by every chromosome. In phase G2, continues protein synthesis and prepares cell for the mitotic phase. There are checkpoints (G1/S or G2/M) to make sure the previous phase is complete prior to embarking on the next step checking DNA. Finally in phase M cell is divided [12].

A series of checkpoints where cyclins and Cyclin Dependent Kinases (CDK) form complexes and in concert allow to control to each phase to Progression the next phase [11]. Mitogenic signals excite cyclin-dependent kinase 4 (CDK4) and CDK6 and encourages the cell division. Initially, complexes CDK4 and CDK6 with cyclin D have a specific preference for the phosphorylation of the tumour suppressor retinoblastoma protein (Rb) which coordinated the function of the family of transcription factors E2F. Whereas the Rb has to be hyper-phosphorylated to proceed to the synthesis step of the cell cycle and DNA replication is initiate. In G1 check point phase, E-CDK2 is added phosphate groups to Rb. So, it becomes gradually phosphorylated, allowing it to be dissociated from E2F that allows E2F to initiate transcriptional program, which contributes to the progress of cells into S phase. Protein kinase inhibitor such as p21 and p27 that inhibit CDK2– cyclin complexes. During the normal cell division, after passing from G1/S start DNA synthesis. CDK1-Cylin A and CDK1-Cyclin B complexes phosphorylate their targets in the G2 stage. In the absence of DNA damage and after adequate preparation for chromosomal segregation will Progress towards mitosis [13]. Mutations in any of these regulatory pathways lead to mutated cells replication or irregular chromosome numbers, contributed in genomic instability [14]. The cell cycle includes several regulatory proteins, tumor-suppressor genes, oncogenes and mitotic proteins for proper cell replication [14].

2-1-1- G1 Checkpoint

When stress is inserted on the cell, p53 as a tumor suppressor gene, code transcription factors in G1-S phase checkpoint and prevent cell division. When DNA damage is observed, the activity of repair enzymes is stimulated at a time. DNA damage is beyond repair, the p53 protein will causes the destruction of the cell [5]. Most observations of the activities of p53 have based on p53 transcriptional sites, p53 protein up regulates endogenous production of p21 protein, which interact with the cell division stimulating cyclin dependent kinase 2 and generate p21- cdk2 complex that cell unable to pass the next level. p53/p21 G1 restricted point have been activated by trauma such as radiation, DNA-damaging chemotherapy, or other stress, such as nutritional deficiency, trigger growth arrest before desirable condition [15].

p21 prevents cyclin-dependent kinase 2 (Cdk2)- complex lead to Rb inactivation, therefore inhibits to entrance cell to S phase .Thus, cell cycle is arrested in the G1 through inhibition Rb-mediated of E2Fdependent transcription [16]. In addition, P21 prevent the binding to E2F to phosphorylated pRB and check the activity of CdK2 [17]. Generally, P21 is recognized as an inhibitor of cyclin-dependent kinase, encoded by the gene CDKN1A situated on chromosome 6p21.2. It triggers cell cycle arrest by identifying with various stimuli and transfer factors such as p53, which by interaction with cell nuclear antigen proliferating, work as a regulator of cell cycle progression and induced G1arrest in cell until cell repair DNA-damage [18,19].

2-1-1- G2 checkpoint

Multiple experiments is established role of p53 in the G2 to M regulation due to the loss of substrates required for DNA synthesis. In cells that have completed DNA synthesis but possess impaired DNA, the G2 checkpoint is activated. Part of the mechanism is restricting cell at the G2/M checkpoint via p53 involves Cdc2 inhibition, which Cyclin-dependent kinase is required to enter mitosis. Three target p53 genes such as p21, Gadd45 and 14-3-3 sigma inhibit Cdc2 [20].

Genotoxic stress often causes p53-dependent pathways that prevent the activation of Cdk2. In human cells, p53-mediated arrest responsible for cell-cycle checkpoint which is critically cyclin B1/Cdk2 complex. When DNA double-strand break by irradiation causes activation of the ATM (Ataxia telangiectasia mutated protein) and ATR (Ataxia telangiectasia and Rad3-related protein) activate the Chk1 and Chk2 kinases (Checkpoint kinase 1 and 2) phosphorylate Cdc25, allowing it to bind to 14-3-3 proteins that anchor Cdc25 in the cytosol where is unable to activate Cdc2 promote arrest of G2/M that prevents cell division [9] [20,21]. Additionally, this modification is produced in 14-3-3δ regulatory protein at the binding site lead to aggregation separated in the cytoplasm and stop replication [21]. Additionally, p53 increase activation and expression of p21, p53-mediated p21 inhibit cyclin B/ Cdk2 prevent progression mitosis and contributes to inactivation of Cdk1 to the G2 checkpoint [22]. In other side, p53 is affected on target gene, a specific 14-3-3δ isoform. 14-3-3s, is up regulated after DNA mutation. Proper localization of nuclear for cyclin B1/CDC2 is inhibited by 14-3-3s. Other the p53 target gene DNA damage-inducible 45 alpha gene (GADD45) interacts with Cdk2 and suppresses its kinase activity, dissociate cyclin B1/ Cdk2 which anchors Cdk2 in the cytosol that cannot induce mitosis. Further, induction of GADD45 consequences increased cytoplasmic cyclin B and in G2 arrest phase [20][23].

p53 inhibits cell cycle arrest at G1 and G2 is delaying cellular disruption sustained from free radicals before entering the post synthesis and process mitosis [10]. With p53’s biochemical functions that promote DNA repair, including nucleotide excision repair and base excision repair, arrested cells will return to the proliferating condition [24].

2-2-Apoptosis

Numerous reports have established the mechanism of p53 in the apoptosis. The cells face p53-induced apoptosis, if the genomic repair cannot completed or the cells no programmed to respond to the stresses of sustainable cell cycle arrest [25]. p53 is activated a large number of genes in various steps of apoptosis signaling and execution during the severe stress [26]. Apoptosis progression is controlled by processes of modulation, the intrinsic pathway also named the mitochondrial pathway and the extrinsic pathway are the two best-understood activation mechanisms [27].

Intracellular signals produced when cells are stressed stimulate the intrinsic pathway and dependent on the release of proteins from the inter-membrane space of the mitochondria. In the other way, extracellular ligands bonding to cell-surface death receptors stimulate extrinsic pathway, which contributes to the death-inducing signaling development [28]. In contrast, the intrinsic pathway is accelerated after the release of cytochrome C from mitochondria [29]. New research suggests that p53 not only controls the permeability of the outer membrane mitochondria, but also promotes inner permeability during oxidative stress [30]. The fundamental mechanism behind the correct action is unclear. There many genes code proapoptotic proteins which are BH3 domain- only (Noxa, Bad, Bax, Puma, Bak, p53AIP1); death receptors (Killer/Dr, Fas, Dr4,); and execution factors for apoptosis (Apaf1, caspase 6, Bnip3L) in mitochondrial membrane. P53 induce the expression genes like Puma, Bad, Bax, Noxa, Bak, and Apaf 1 target multiple stages of the permeability of the outer membrane mitochondria regulatory mechanism facilitate the apoptosis [31].

p53 induces activation of Fas and Dr5, stimulates the mechanism of extrinsic apoptosis through facilitating death receptor dimerization such as procaspase 8 expression and executioner caspase 3 and 7 activation, then eliminate destroyed cells. In intrinsic apoptosis pathway in the severe damage, P53 causes the activation of the BH3-only proteins triggers permeability of the mitochondrial outer membrane, which is a key step in the intrinsic apoptosis pathway that promote the mitochondrial membrane potential disturbance and cytochrome c release result apoptosome complex formation. Apoptosomes recruit and activate procaspase 9, which further triggers executioner caspase 3 and 7 then finally cell death [31-33].

P53 proteins fusion to a signal peptide in the mitochondria is enough to induce apoptosis and does not involve activation of transcription factors. Biochemically, P53 will attach to Bcl2 and BclXL, encouraging Bax and Bak release and activation which are pro apoptotic proteins that incorporated into the outer membrane, and oligomerzed to form pore to release cytochrome c as well as other proteins such as Smac and Omi from the inter membrane. Cytochrome c attaches to adenosine triphosphate (ATP) and Apaf1, facilitating oligomerization of Apaf1 to create an apoptosome complex. The apoptosome employs and stimulates procaspase 9, which further activates caspase 3 and 7 to cause cell death [31].

2-3 - Senescence

Cellular senescence is characterized as a cell state with prolong and irreversible cell-cycle arrest and the acquisition of various phenotypic modifications, including morphological changes, chromatin modifications, metabolic pathway alteration and induction of pro- inflammatory factors or (senescence associated secretory phenotypes) SASP [34]. Recently it is estimated by cytokines, proteases, growth factors, and non-soluble extracellular matrix proteins [33]. All these features is essentially restricted the proliferation of both old and impaired cells relate to a normal cell cycle under physiological conditions [35]. Senescence has been correlated with incremental telomere shortening, a physiological function that happens any time the cell divides and impairs the genomic integrity and stabilization associated with the activation of particular molecular senescence pathways [36,37].

p53 plays a crucial role and can be triggered in a DNA-damage response (DDR) through DDR-dependent or DDR-independent way in the form of senescence [38,39]. In the first case, telomere erosion, DNA damage, as well as hyper activation of oncogenes and inactivation of onco-suppressors resulting from replicative stress activate the DNA damage repair cascade [40]. DDR stimulates the ataxia telangiectasia-muted (ATM) or ataxia telangiectasia Rad3-related kinases of the tension sensors (ATR). In exchange, ATM/ATR stimulates the p53/p21 axis by phosphorylation of both p53 and its ubiquitin ligase Mdm2, which results stabilization [41].

Several internal or external stress factors activate DDR pathway, which activates the p53 and/or p16 pathways in turn. P16 inactivates Cdk4/6 leads to the aggregation of pRb, stops the modulation of transcription factors of E2F and drives cell cycle arrest or senescence [42,43]. SO, the severity and duration of the stress stimulation seem to be a major factor in the induction of senescence; senescence appears to involve a stable stimulus, while temporary stimuli only cause a temporary growth arrest, encouraging the cell to attempt to restore the damage. Most serious stimuli contribute to apoptosis respectively [44].

3- Role of p53 in progressing of Cancer

P53 is one of the tumor suppressor proteins most investigated, with mutations that contribute to loss of wild type p53 function often detected in several various forms of tumor [45]. When tumor suppressor genes like p53 are mutated, the cells are unable to respond to cell-cycle checkpoints normally and DNA damage is severe unable to trigger programmed cell death. This could contribute to a more increase in abnormalities and the inability of the infected cell to escape the cell cycle as it may become umorigenic [46].

The mutations of the p53 include frame shift, missense and deletion missense is estimated about 74% which a single substitute of the original amino acid with other substitute one and mostly happen in the DNA-binding domain. On this domain many hotspot mutations like as Arg-175, Arg-249, Arg-273, Arg-282, Gly-245, Tyr-220

and Arg-248 is identified [47]. The frequency of TP53 mutation in hematopoietic malignancies, the prevalence of TP53 mutations ranges from ~10% to 50-70% in vaginal, colorectal, and head and neck malignancies [48]. Li-Fraumeni syndrome, which is a family cancer syndrome like breast cancer, soft tissue sarcoma, and several other forms of cancer induced germline mutation of P53 [49]. Several P53 mutations are found in the DNA-binding domain, thereby preventing p53 from transcription its target genes. However, mutant p53 has led to a loss of normal function and also enhanced tumor potential [48]. Defects in TP53 causes impart cancer vulnerability can be hereditary or somatic.P53 germline defects cause Li-Fraumeni syndrome which cause a number of early-onset malignancies to be predisposed [50]. In several tumors, mutant p53 proteins are highly expressed and lead to progress tumor growth and drug resistance through inhibiting wild type p53 member [51]. A majority of mutant p53 function originates from ability of mutant p53 to manipulate gene transcription. It has been shown that mutant p53 transcriptional effects contribute to increased cellular, apoptosis resistance that can be induced by mutant p53 association with Ets-2, increased of migration, invasion, tumor inflammation, and increased metastasis [52].

In conclusion, P53 protein activate and transcripted of an important number of direct target genes in response to cellular stress such as DNA damage. This responses depend on intensity of the stress, include in cell cycle arrest in G1 or G2, DNA repair, apoptosis (intrinsic and extrinsic pathway),Senescence (DDR-dependent or DDR-independent). In addition, P53 mutations has been correlated with as an oncogene expression in many cancer types.

Acknowledgments

I would like to thanks and appreciates the Professor Dr. Alaa Hani Raziq- pathologist in College of duhok Medical University for his kind help for this review.