Structure and Function of p53 

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Discovery of p53, its Initial Characterisation as a Proto-oncogene, and Subsequent Studies that Corrected for that Mischaracterisation and Assigned p53 the Role of a Tumour Suppressor

In a study by Lane & Crawford (1979), the large T antigen of the SV40 virus was found to form a complex with a particular protein coded for by the cell in SV40-transformed mouse cells. This observation was made by immunoprecipitating an extract of SVA31E7 SV40-transformed mouse cells with rabbit antiserum specific for the large T antigen. That cell-encoded protein is currently known as p53, the well-studied protein product of the tumour suppressor gene TP53, which is the most mutated gene in human cancers that has been identified so far (Nigro et al., 1989).  The association between p53 overproduction and malignant transformation initially led Eliyahu et al. (1984) to assign an oncogenic role to p53, as they found that rat embryo fibroblasts transfected with both p53 and active Ha-ras were induced to undergo malignant transformation, which manifested in the formation of foci of distinct morphology that were detectable after 9-12 days. This observation only occurred in rat embryo fibroblasts that were co-transfected with both p53 and active Ha-ras, as p53 or Ha-ras alone were insufficient for the induction of foci formation.

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It was later found that rat embryo fibroblasts that were co-transfected with wild-type p53- and ras-expressing DNA clones formed a significantly reduced number of transformed foci, when compared to the number formed by cells co-transfected with mutant p53 and ras. Furthermore, transformed foci were nearly undetectable in those cultures that were transfected with ras, mutant p53 and wt-p53 simultaneously. Similarly, cultures co-transfected with ras, E1A and wt-p53 formed 67-80% less transformed foci on average than cultures co-transfected with ras and E1A alone (Finlay et al., 1989). These results indicated that, contrary to the conclusion reached by Eliyahu et al. (1984), p53 should be classified as a recessive oncogene, or tumour suppressor, rather than a proto-oncogene (Finlay et al., 1989; Eliyahu et al., 1989).

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Functioning as a tumour suppressor, p53 manifests as a multifunctional nuclear phosphoprotein characterised by discrete structured and unstructured domains (Joerger & Fersht, 2007 : Römer et al., 2006). Bell, Klein, et al. (2002) postulated that p53 is a member of the expanding cohort of loosely folded or partially unstructured native proteins. Approximately 37% of its constitution is inherently unstructured (Iakoucheva et al., 2002). The absence of a rigid conformation, coupled with its comparatively diminished overall stability, facilitates the physiologically versatile interaction of p53 with an array of partner proteins and the modulation of its turnover (Bell, Klein, et al., 2002). Additionally, p53 assumes a homotetramer configuration (four identical monomers), whereby each monomeric subunit consists of 393 amino acid residues and approximately weighs  43 kDa (Dawson et al., 2003 ; Nigro et al., 1989). 

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The acquisition of a c-DNA clone derived from F9 embryonal carcinoma cells containing the complete sequence information for murine p53 protein revealed that the protein consists of three distinct domains: (i) an N-terminal domain (NTD) consisting of the first 75 amino acids, characterised by an abundance of acidic amino acid residues such as Asp and Glu (Pennica et al., 1984). The NTD includes the transcriptional activation domain (TAD) (residues 1-73) and a proline-rich domain (PRD) (residues 61-94) (Fields & Jang, 1990 : Walker & Levine , 1996); (ii) a central, hydrophobic, sequence specific DNA-binding domain (DBD) spanning residues 102-292 (Pavletich et al., 1993; Bargonetti et al., 1993), and (iii) a C-terminal domain (CTD) comprising of the tetramerization domain (TD) (residues 315-350) that also forms part of the non-specific DNA-binding region ranging from residues 315-390 and a regulatory domain (residues 363-393) (Hupp et al., 1992; Gu & Roeder, 1997 : Wang et al., 1994; Wang et al., 1993). 

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Figure 1: P53 monomer. The TAD is highlighted in dark red and red. The proline-rich region is highlighted in red and yellow. The TAD and the proline-rich region are highlighted in red. The sequence-specific DNA-binding domain is highlighted in orange. The tetramerization domain is highlighted in green. The non-specific DNA-binding region is highlighted in green, blue and cyan. The regulatory domain is highlighted in cyan and magenta. The non-specific DNA-binding region and the regulatory domain are highlighted in cyan. The remaining sequences are highlighted in grey.

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Transactivation Domain (TAD)

The transactivation domain that was identified by Fields & Jang (1990) to consist of the N-terminal 73 amino acids was later found to consist of two subdomains: the first, consisting of  residues 1-42, was found to transactivate in a manner comparable to wild-type p53, whereas the second subdomain was found to consist of residues 40-83 (Unger et al., 1992; Candau et al., 1997). One means of p53 carrying out its effects of target gene transcription is by interacting with various components of the transcription machinery (Seto et al., 1992; Liu et al., 1993; Thut et al., 1995; Lu & Levine, 1995).

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The leucine 22 and Tryptophan 23 hydrophobic residues were found to be crucial for p53’s interactions with the transcription machinery and with p53-responsive elements of target genes whose transcription it promotes, as mutations at these positions abolished p53’s transcription factor capabilities (Lin et al., 1994). In addition to these two residues, the tryptophan 53 and phenylalanine 54 residues in the second subdomain were also found to be critical for transactivation activity (Candau et al., 1997).

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P53 influenced target gene transcription by interacting with components of the transcription machinery, such as the TATA-binding protein (TBP) within the TFIID complex. Notably, residues 20-57 in p53’s transactivation domain interacted with TBP residues 220-273. Mutations at positions 22 and 23 in p53 impaired transcriptional capabilities (Seto et al., 1992; Liu et al., 1993).  

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p53 also interacted with TBP-associated factors (TAFs), such as hTAFII31, a subunit of TFIID. Studies showed that fusion proteins of GST and p53 regions including the amino terminal 92 residues bind  hTAFII31, and mutations at positions 22 and 23 impaired this interaction (Lu & Levine, 1995). Moreover, Thut et al. (1995) identified interactions between p53's first 42 amino acids and two TFIID components, dTAFII40/hTAFII32 and dTAFII60/hTAFII70, which were lost with alanine substitutions at residues 22 and 23. Partial TFIID assemblages containing TAFII60 or a complex of TAFII40 and TAFII60 were sufficient for p53 activation domain-dependent transcriptional activation. These findings contribute to understanding the intricate molecular interactions underlying p53-mediated transcriptional regulation.

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Proline-Rich Region 

The proline-rich region spanning residues 61-94 was identified by Walker & Levine (1996) and was found to contain five repeats of the PXXP motif, with P representing proline and X representing any amino acid. Despite this region being localised between the sequence-specific DNA-binding region and the N-terminal transactivation domain, a deletion mutant lacking residues 62-91 of the proline-rich region was not found to interfere with p53’s transactivation capabilities, indicating that it is not crucial to the functioning of either of these other two domains. However, its deletion compromised p53’s growth suppressive activity, suggesting a role for this region in tumour suppression mediated by p53. Furthermore, the PXXP motif was found to contribute to the apoptotic functions of p53 (Venot et al., 1998). 

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Core DNA-binding Domain 

The core DNA-binding domain was localised to a central region of p53 spanning residues 102-292 and was found to be responsible for sequence-specific DNA binding (Pavletich et al., 1993; Bargonetti et al.,1993; Kern et al 1991). The crystal structure of the core DNA-binding domain with a consensus DNA-binding site was obtained using crystals containing one DNA duplex and three p53 core DNA-binding domains in an asymmetric unit. 

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Figure 2: p53’s core DNA-Binding Domain bound to a 21-bp DNA duplex (PDB:1TSR). This crystal structure was determined by Cho et al. (1994).

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The DNA-binding domain was found to consist of: a 2 antiparallel  β-sheet sandwich, with the  β-sheets consisting of 4 and 5  β-strands respectively; a loop-sheet-helix motif that was tightly packed against one end of the  β-sandwich; and a tetrahedrally coordinated zinc atom holding two large loops together at this same end of the  β-sandwich. In spite of the β-sandwich forming a substantial proportion of the DNA-binding domain, it was not found to be directly involved in DNA-binding, unlike the loop-sheet-helix motif and one of the 2 large loops. In fact, structures involved in DNA-binding were found to be the conserved regions of the domain that are hotspots for mutations in tumours (Cho et al., 1994).  

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Pavletich et al. (1993) also carried out proteolytic digestion experiments that revealed the presence of a 53-residue structural domain within the C-terminal region spanning residues 311-363, followed by a flexible linker and a highly basic region consisting of residues 368-387. Their analysis of peptides constituting this region indicated that the 53-residue structural domain (residues 311-363) is responsible for tetramerization, whereas the 20-residue basic region (residues 368-387) contributes to non-specific DNA binding.

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Figure 3: A schematic representation of the amino acid residues assigned to the core DNA-binding domain, tetramerization domain and the non-specific DNA-binding domain by Pavletich et al.  (1993). The sequence specificity of the core DNA-binding domain was determined by Kern et al. (1991).

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Tetramerization Domain 

In agreement with Pavletich et al. (1993), Wang et al. (1993) found that p53 contains two autonomous DNA-binding domains (residues 102-292). One of the domains was found to possess both sequence-specific and nonspecific DNA-binding ability, whereas the other domain bound DNA non-specifically. The domains were localised to the regions spanning residues 80-290 and 315-390, respectively, with a loss of DNA-binding function occurring upon shortening these regions by 30-35 residues from either end. Moreover, amino acids 1-290 were found to be sufficient for p53-mediated transactivation of a p53-specific in vivo promoter, indicating that stable tetramerization is not required for the transactivation and sequence-specific DNA-binding p53 functions, since residues 311-363 were found to be responsible for tetramerization in Pavletich et al. (1993)’s study.

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Figure 4: A schematic representation of the amino acid residues assigned to the specific and nonspecific core DNA-binding domain and the non-specific DNA-binding domain by Wang et al.  (1993). 

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Wang et al. (1994) later found that murine p53 contains two autonomous oligomerization domains spanning residues 315-350 and 80-320. The former was found to form stable tetramers and so was determined to be the core tetramerization domain. This region is similar to the 53-residue region identified by Pavletich et al. (1993) as having a role in tetramerization. On the other hand, the latter oligomerization domain was determined to be weak in relation to the 315-350 region. The location of this weaker oligomerization domain indicates a potential role for it in p53 binding to DNA consensus sequences supported by evidence obtained by means of scanning transmission electron microscopy.

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Figure 5: A schematic representation of the amino acid residues assigned to the weak oligomerization and strong tetramerization domains  by Wang et al.  (1994). 

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Figure 6: This is a summary of the above text and figures showing an agreement between the results of the above studies (Pavletich et al., 1993; Wang et al., 1993; Wang et al., 1994).

 

The tetramerization of p53 was found to have a role in the protein’s subcellular localisation, as a nuclear export signal (NES) rich in leucine was found in the tetramerization domain, with modification of its primary amino acid sequence influencing both tetramerization and nuclear export of p53. p53 in neuroblastoma cells, which normally contain cytoplasmic p53 due to defects in nuclear retention and hyperactive export, was successfully retained in the nucleus upon binding a p53 tetramerization domain peptide that contains the NES.  These results along with the NES’ conserved localization within the tetramerization domain indicates a role for tetramerization in the nuclear localization and activation of p53 by covering the NES and making it inaccessible to nuclear export machinery, particularly the CARM1 export receptor (Stommel et al., 1999). 

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Regulatory Domain 

The C-terminal domain of p53 was determined to be involved in the regulation of the protein’s DNA-binding activity. The removal of the C-terminal 30 amino acids of p53 resulted in constitutive DNA binding in a multimeric form, indicating that this 30-residue region has a regulatory role in p53’s DNA binding. Modifications that presumably interfere with the activity of the C-terminal amino acid motif, were found to be capable of activating p53 DNA binding. Such modifications include phosphorylation by casein kinase II, interaction with DnaK, and trypsin-mediated proteolytic cleavage (Hupp et al., 1992). 

 

The effect of the C-terminal regulatory domain was further characterised by Retzlaff et al. (2013), who found that an intermolecular mechanism, in which the regulatory domain of one p53 monomer interacts with the DNA-binding domain of another p53 monomer, stabilises the p53 tetramer. Upon encountering a DNA binding site, the DNA binding domain’s higher affinity for DNA results in the bound regulatory domain being displaced by DNA. The released C-terminal domain can then become engaged in non-specific interactions with DNA.

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Figure 7: Domain Organization of p53. P53 can be dissected into three major parts: the NTD (NTD; amino acid residues 1-95), the sequence-specific DNA-binding core-domain (DBD; 102-292); and the C-terminal domain (CTD; 292-393). The NTD is subdivided into the transcription activation subdomain (Pro; 61-94), the CTD is subdivided in the tetramerization domain (TD; 315-350), and the regulatory domain (CTRD; 363-393).

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Isoforms of p53

Mutations have traditionally been regarded as the primary mechanism leading to p53 inactivation, however, recent studies have uncovered the significant presence of p53 isoforms in various cancer tissues, suggesting potential roles in carcinogenesis (Vieler & Sanyal, 2018). The expression of p53 isoforms is governed by alternative splicing and alternative promoters (Khoury & Bourdon, 2009), playing crucial roles in coordinating cellular responses under physiological conditions and influencing the p53 pathway in conjunction with full-length p53 (Flp53) (Hafsi et al., 2013; Slatter et al., 2015). Characterizing isoforms and understanding their functions is essential for cancer prognosis and in the field of cancer biology (Vieler & Sanyal, 2018).

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The TP53 gene, referred to as a bifunctional gene due to two functionally defined promoters (P1 and P2) (Khoury & Bourdon, 2009), can generate twelve distinct p53 isoforms through alternative splicing, various promoters, and two internal expression entry site (IRES) motifs (Khoury & Bourdon, 2009 ; Vieler & Sanyal, 2018). Alternative promoters, P1 and P2, transcribe TP53 gene, yielding multiple mRNA transcripts (Bourdon et al., 2005). Additionally, alternative splicing at intron 2 or intron 9 result in N- and C-terminally shortened variants (Arai et al., 1986; Horvat et al., 2021). The TP53 gene has 11 exons, including alternative exons 9a and 9b, and intron 2 (i2) and 4 (i4). Furthermore, TP53 mRNA translation may begin at several codons, most notably at positions 1, 40, 133, and 160, leading in the formation of different p53 isoforms (Joruiz & Bourdon, 2016; Anbarasan & Bourdon, 2019). 

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Figure 8: A schematic representation of TP53 and p53 isoforms. (A) FLp53 gene structure consisting of 11 exons (shown as coloured boxes), two alternative exons (9 and 9) and introns 2 (i2) and 4 (i4) (shown as striped boxes). Two alternative promoters have the ability to transcribe the TP53 gene. (B) Modular structure of the p53 isoforms with their respective functional domains (shown in different colours). 

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The p53 isoforms, categorized as Δ160p53α/β/γ, Δ133p53 α/β/γ, Δ40p53 α/β/γ, and p53α/β/γ, exhibit deficits near the N- and/or C-termini compared to full-length p53 (FLp53). N-terminally truncated isoforms, Δ40p53, Δ133p53, and Δ160p53, lack the first 39, 132, or 159 residues, respectively. In Δ133p53 and Δ160p53, the absence of the first 132 and 159 residues leads to the loss of both TAD1 and TAD2, along with the proline-rich domain (PRD). These isoforms can be further truncated at the C-terminus, with the α, β, and γ designation indicating the degree of C-terminal truncation. In the case of α-isoforms exons 10 and 11, which encode for the Oligomerization Domain (OD) and C-terminal Domain (CTD), are maintained; however, in the β and γ isoforms, the presence of premature termination codons (PTCs) in exons 9β and 9γ results in the absence of both the OD and CTD (Joruiz & Bourdon, 2016; Anbarasan & Bourdon, 2019).

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Roles of p53 

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DNA Damage Response

Under normal conditions, p53 levels remain low due to its short half-life and predominantly inactive state, rendering it ineffective in DNA binding and transcription activation. 

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In 1991, Kastan et al. identified a role for p53 in the cellular response to DNA damage, as they observed a rise in p53 levels in both ML-1 myeloblastic leukaemia cells and normal, proliferating human bone marrow progenitor cells following the induction of DNA damage by means of γ-irradiation (XRT). In addition to p53 accumulation, ML-1 cells that were exposed to non-lethal doses of XRT also exhibited a transient reduction in cells undergoing DNA synthesis in the S-phase of the cell cycle, with this decrease being due to cells becoming arrested primarily at the G1-phase after exposure to low doses of XRT.  On the other hand, G2/M arrest was found to increase in prominence at higher doses. The accumulation of p53 and the cell cycle arrest were only found to be well correlated in cells with intact p53 genes. This indicates a role for wild-type p53 in the transient inhibition of DNA synthesis by means of G1 cell cycle arrest following DNA damage.

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This notion was further supported by the finding that abnormal cell cycle responses to DNA damage can occur as a result of the loss of wild-type p53 expression, or the overexpression of mutant p53, resulting in such cells proceeding to S-phase despite their damaged DNA templates and producing genetically unstable daughter cells. Conversely, G2 arrest was found to have occurred in all cell types following DNA damage, regardless of their p53 gene status (Kastan et al., 1991).

 

The above findings were explored further by Kastan et al. (1992). In this study, the defective genes that result in the autosomal recessive, genome instability syndrome, Ataxia-telangiectasia (AT), were found to be required for an increase in the levels of p53 protein following exposure to ionising radiation (IR), which, in turn, is required for the induction of a Growth Arrest and DNA Damage-inducible (GADD) gene, GADD45. In fact, AT cells were found to be deficient in GADD45 expression following IR, and wild-type p53 was found to bind a conserved intronic sequence within the GADD45 gene. This suggests that the cell recognises IR-induced DNA damage and responds by increasing p53 protein levels in a manner involving AT gene products. Following p53 protein accumulation, p53 upregulates the transcription of the GADD45 gene, whose mRNA levels are normally low, and triggers cell cycle arrest at G1.

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Though insights into p53’s role in the cell’s response to DNA damage were being uncovered, the mechanism allowing for the recognition of the presence of DNA damage by p53 remained unknown. In 1994, Nelson and Kastan studied various genotoxic agents’ induction of p53 and identified DNA strand breaks as the DNA lesions that are critical for inducing the accumulation of the p53 protein. P53 protein accumulation following the formation of DNA photoproducts as a result of exposure to UV radiation was found to occur as a result of the DNA strand breaks that form during replication of such damaged DNA or during the excision repair process. In addition to DNA strand breaks, insertion/deletion mismatches were also found to be recognised and tightly bound by p53, predominantly in tetrameric form, by means of its 14-kDa C-terminal domain. The formation of such a stable p53 complex at a DNA lesion site could be suggestive of p53’s role as a scaffold for the assembly of DNA repair, multiprotein complexes (Lee et al., 1995).   

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DNA Repair

The GADD45 gene whose expression was found to be upregulated by p53 in response to DNA damage (Kastan et al. 1992) was investigated for its role in growth control and DNA repair by Smith et al. (1994). In this study GADD45 was found to interact with Proliferating Cell Nuclear Antigen (PCNA). The suggested mechanism for how this GADD45-PCNA interaction contributes to DNA repair is as follows: Following DNA damage, GADD45 expression is upregulated in a p53-dependent manner. The GADD45 proteins, now present in a higher quantity than prior to the DNA damage event, interact with free PCNA or recruit PCNA from other complexes, such as cyclin-dependent kinase or replication complexes, resulting in growth inhibition. PCNA, which is required for in vitro nucleotide excision repair and acts as a DNA polymerase δ and ε auxiliary factor, can then be redirected to participate in DNA repair (Smith et al., 1994; Shivji et al., 1992). In addition to facilitating the formation of GADD45-PCNA interactions by upregulating GADD45 expression in response to DNA damage (Kastan et al., 1992; Smith et al., 1994), p53 was also found to influence nucleotide excision repair by means of its C-terminal domain associating with TFIIH subunits with a potential role  in the process, namely XPB, XPD, the yeast homologue of XPD (Rad3) and CSB (Wang et al., 1995). 

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Offer et al. (1999)’s study further expanded p53’s role in DNA repair by showing that p53 may be involved in base excision repair. Temperature-sensitive murine and human p53 mutants were found to enhance DNA repair synthesis in nuclear extracts maintained at permissive temperature in vitro. P53 with a wild-type conformation was also found to induce base excision repair activity within a cell-free system. Conversely, mutant p53 did not induce base excision repair. Instead, it seemed to interfere with p53-independent base excision repair activity.

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p53 Regulation

The importance of p53 in the cell cannot be understated. In order for the protein to achieve its function, many crucial biomolecules are required to regulate the cell cycle in order to ensure the cell may undergo cell division in a healthy and safe manner. In order to achieve this the regulation of p53 is maintained through two distinct pathways joined together by (ADP ribosylation factors) ARF which is a multifunctional protein which regulates the p53 pathway in a negative manner (Ewen et al., 1993 ; Kubbutat et al., 1997 ; Hermeking et al., 1997). 

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The first pathway relates to the direct regulation of p53 protein levels. This occurs in two scenarios: detection of DNA damage or inappropriate mitogenic signalling. When one or both of these conditions are met, p53 lifespan is increased through the production of p14ARF or p16INK4a. The increased life span allows for accumulation p53 and results in cell death. The effect of p14ARF and p16INK4a  transcription is inhibited by MDM2. This protein is responsible for reducing p53 lifespan in the cell preventing it from working. (Hermeking et al., 1997 ; Kubbutat et al., 1997).
 

The retinoblastoma (RB) protein works hand in hand with p53 as they both regulate the same pathway through negative feedback. For DNA synthesis to occur RB is phosphorylated by cyclin dependent-kinases (CDK) during the G1 phase of the cell cycle. The inhibitory CDK is produced when stimulated by Ras in the presence of abnormal mitogenic signals (Ewen et al., 1993). When this occurs, RB does not associate with histone deacetylase and E2F transcription factors preventing gene expression leading to DNA synthesis. p16INK4a works in tandem with RB. When needed this protein is capable of inhibiting the CDKs preventing E2F activation. As a result cells are blocked from exiting the G1 phase (Hermeking et al., 1997).

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When oncogene activation occurs, E2F and Myc are often overexpressed. To counteract this, ARF expression is triggered, causing activation of p53 through inhibiting MDM2. This results in cell-cycle arrest or apoptosis. (Ewen et al., 1993; Hermeking et al., 1997).

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Figure 9: Diagram of p53 Pathway - This figure illustrates the various proteins involved in the regulation of p53. The pathway is made up of two distinct pathways with the Ras/RB protein at the top and the p53/MDM2 pathway at the bottom joined together at ARF.

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P53’s Role in Apoptosis 

p53 plays a crucial role in regulating apoptosis. The N-terminal transcriptional transactivation domain, DNA-binding domain, and tetramerization domain collaboratively activate the transcription of various p53 target genes, including pro-apoptotic proteins. The acidic N-transactivation domain is essential for gene activation, while the core DNA-binding domain enables sequence-specific DNA attachment. The tetramerization region facilitates the formation of p53 dimers and tetramers, necessary for favourable gene expression regulation (Hardin et al., 2011). 

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Under normal conditions, p53 levels remain low due to its short half-life and predominantly inactive state, rendering it ineffective in DNA binding and transcription activation. Cellular stresses such as DNA damage, hypoxia, and nucleotide deprivation trigger p53 activation. DNA damage induced by factors like ionizing radiation, radio-mimetic drugs, ultraviolet light, and chemicals activates the ataxia-telangiectasia-mutated (ATM) protein kinase. Activated ATM phosphorylates p53, inhibiting its interaction with Mdm2. Accumulated p53 orchestrates cell cycle arrest and cell death. It acts as a transcription factor for specific genes, including p31, which blocks cell cycle progression, and enzymes involved in DNA repair. If the damage is irreparable, p53 activates genes, including PUMA (p53 upregulated modulator of apoptosis), initiating apoptosis by deactivating the apoptosis inhibitor Bcl-2 (Hardin et al., 2011).  

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p53 serves as a molecular gatekeeper, preventing cells with damaged DNA from reproducing and passing on the damage. Dysregulation in cell cycle control contributes to the unrestrained growth observed in cancer cells. p53’s role in initiating cell cycle arrest  and cell death is crucial for preventing the reproduction of cells with damaged DNA. Cancer cells, exhibiting malfunctioning cell cycle controls and unresponsiveness to internal conditions like DNA damage, proliferating uncontrollably, leading to the aberrant behaviour observed in cancerous growth. Understanding these intricate mechanisms provide insights into potential therapeutic strategies targeting p53 in cancer treatment (Hardin et al., 2011). 

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Figure 10: Role of the p53 Protein in Responding to DNA Damage. Damaged DNA activates the ATM or ATR protein kinase, leading to activation of checkpoint kinases, which leads to phosphorylation of the p53 protein. Phosphorylation stabilizes p53 by blocking its interaction with MDM2, a protein that would otherwise mark p53 for degradation. When the interaction between p53 and Mdm2 is blocked by p53 phosphorylation, the phosphorylated p53 protein accumulates and triggers two events. (1) The p53 protein binds to DNA and activates transcription of the gene coding for the p21 protein, a Cdk inhibitor. The resulting inhibition of Cdk-cyclin prevents phosphorylation of the Rb protein, leading to cell cycle arrest at the restriciton point. (2) When the DNA damage cannot be repaired, p53 then activates gene coding for a group of proteins that trigger cell death by apoptosis. A key protein is PUMA, which promotes apoptosis by binding to, and blocking the action of, the apoptosis inhibitor, Bcl-2. 

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p53 in Autophagy

Autophagy is the process which regulates the delivery of cytoplasmic constituents to the lysosome for degradation. This process is essential for the cell in order to monitor the integrity of proteins and organelles. The rate at which degradation occurs varies depending on the specific cargo or stress. Autophagy also has a special relationship with p53 and many studies have illustrated the dynamic relationship between the two. This dynamic is especially important when studying cancer as autophagy may be used to suppress p53 production thus promoting tumour progression and prevent tissue degradation (Guo et al., 2013 ; Mizushima and Komatsu 2011).

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Autophagy is regulated via the autophagy-related genes (Atgs). The products of these genes oversee the formation of double-membrane vesicles which bind and capture intracellular components. Once degraded, the products are released into the cytoplasm where they are then recycled into various biochemical pathways (Mizushima and Komatsu 2011).

 

The mTOR/AKT is a biochemical pathway which regulates autophagy based on nutrient availability and stress. mTOR is a protein which regulates autophagy, protein synthesis and metabolism. Starvation and loss of mTOR signalling results in phosphorylation of various autophagy repression proteins. As these proteins are inactivated, AMP-activated protein kinase (AMPK) is activated which enables autophagy to take place at the transcriptional and posttranscriptional level. Once this occurs, energy metabolism, energy homeostasis and cell survival are restored. In hypoxic conditions hypoxia-inducible factors (HIFs) are employed to transcriptionally activate Atgs and autophagy in order to ensure cell survival in hypoxic conditions (Bunz et al., 1999). As previously mentioned, these pathways are regulated by p53 in the same manner through a variety of mechanisms such as gene transcription or nontranscriptional mechanisms in order to either aid in stress adaptation (e.g., cell-cycle arrest) or to eliminate cells that are beyond repair by apoptosis or senescence (Ocana et al., 2014). 

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Studies using mouse embryofibroblasts have demonstrated that Atg7 deficiency increased p53-dependent apoptosis (Lee et al. 2012). Autophagy-deficient mice also die during physiological neonatal starvation. Force-feeding these mice only delays death. Despite these studies, it is still unclear whether neonatal death of autophagy-deficient mice is strictly dependant on p53 (Guo et al., 2013).

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In cancer cells, autophagy suppresses p53 which results in tumorigenesis (Guo et al., 2013). A mouse model study of hereditary breast cancer caused by loss of the (Partner And Localizer of BRCA2) Palb2 tumour suppressor which works with the BRCA2 protein to fix damaged DNA and Atg6/Beclin1 a mammalian protein which is responsible for autophagic induced apoptosis, resulted in a suppression of tumorigenesis and an increased life-span. This is because the beclin1 deficiency is compensated for by compound p53 deficiency. As DNA damage and oxidative stress occur due to Palb2 deficiency, p53 is activated. This is further enhanced when autophagy is compromised (Huo et al. 2013).

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Figure 11: Role of p53 in Autophagy - This figure shows the various biochemical machinery responsible for the process of autophagy. In the presence of DNA damage or cellular stress, p53 binds to DNA inducing the expression of various proteins. This triggers the formation of the autophagosome and autolysosome which results in biomolecule degradation and recycling.

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The Role of p53 in Metabolism

The metabolism of various biomolecules is at the core of tumour growth and malignant transformation for example tumours have a tendency to drastically increase glucose uptake and glycolysis to produce sufficient energy for cellular division. As such the study of how these pathways work and how they are regulated in the cancer cell may be a vital component in combating the disease (Schwartzenberg-Bar-Yoseph et al., 2004). 

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In essence metabolism is divided into two distinct states depending on nutrient availability. If nutrients are abundant, the synthesis of lipids, proteins and nucleic acids occurs and cell growth and proliferation occur. If nutrients are scarce or unavailable, a series of events are triggered in the cell in which energy production is favoured over proliferation and existing stores of macromolecules are broken down  (Ocana et al., 2014). 

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The mTOR protein is regulated by AKT. This molecule promotes anabolic, energy-intensive pathways and is induced in the presence of growth factors to activate mTOR (Bunz et al., 1999). AMP-activated protein kinase (AMPK) on the other hand inactivates mTOR. AMPK expression is increased when the AMP/ATP ratio is high and promotes energy producing catabolic pathways. In this low energy state, mTOR is suppressed. This pathway is connected to many metabolic processes which regulate biomolecule synthesis and energy production (Ocana et al., 2014). 

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This metabolic switch is further regulated via p53 activation when nutrients are scarce. Once AMPK is activated and AKT is inhibited, p53 further stimulates AMPK production downstream via activating sestrin and tuberous sclerosis 2 (TSC2). This results in the inactivation of mTOR and reduced cell proliferation (Leconte et al., 2011). 

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Remarkably, p53 is also capable of slowing down the glycolysis and oxidative phosphorylation pathways, countering the dramatic increase of these pathways which is typical in cancer. Studies have shown that p53 can reduce glucose uptake in the cell via inhibiting gene expression of GLUT1 and GLUT4 transporters (Schwartzenberg-Bar-Yoseph et al., 2004). Not only can p53 reduce glucose uptake but it also has a direct effect on reducing the glycolysis pathway itself as it may decrease levels of phosphoglucomutase. TIGAR is another enzyme that inhibits glycolysis in response to p53 and accomplishes this through lowering the levels of fructose-2,6-bisphosphate (Bensaad et al., 2006). 

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p53 is also needed in oxidative phosphorylation as its many functions have a profound influence on the pathway. Such effects include the transcriptional activation of subunit I of cytochrome c oxidase52; activation of expression of synthesis of cytochrome c oxidase 2 (sCO2)52, which is a key regulator of the cytochrome c oxidase complex; and the induction of expression of the ribonucleotide reductase subunit p52R2, a protein that contributes to the maintenance of mitochondrial DNA (de Lonlay et al., 2007).

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Figure 12: Effect of p53 on Glycolysis - This diagram illustrates the effect of p53 on glycolysis. As p53 reduces GLUT1 and GLUT4 levels, glucose uptake is reduced and glycolysis is subsequently slowed. p53 also reduces phopshoglyceromutase levels and induces TIGAR gene expression which further slow the process down. 

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p53 in Disease 

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Cancer

Due to its role as a tumour suppressor gene, it is no surprise that somatic mutations in TP53 gene are present in roughly 50% of all human cancers, with its frequency varying between different tumour types, reaching as high as 60% in ovarian and colorectal cancers (Royds & Iacopetta, 2006). Upon mutation, wild-type p53 loses its tumour suppressive functionality, and instead, becomes overexpressed acquiring oncogenic properties that further aid tumour progression, termed “gain of function” (Alvarado-Ortiz et al., 2023). In different forms of cancer, p53 fulfils different clinical roles: for example, in colorectal cancer, mutations in TP53 gene have been seen to be one of the more common genetic mutations that lead to the development of this disease (Rodrigues et al., 1990). In breast cancer on the other hand, the accumulation of p53 has been suggested as an independent marker of predicting cancer patient survival rates and disease recurrence (Petitjean et al., 2007). 

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Li-Fraumeni Syndrome

Whilst the above subsection described the somatic mutations in TP53, germline mutations also exist. These are primarily found as missense mutation, leading to Li-Fraumeni Syndrome (LFS), a hereditary heterogeneous autosomal dominant disorder which increases the familial predisposition to cancer (Li & Fraumeni, 1969; Varley, 2003). Families with LFS have a 90% chance of developing cancer throughout their lifetimes, as well as tend to present with rarer forms of cancer, such as sarcomas, brain tumours, adrenocortical carcinomas, premenopausal breast tumours and leukaemia (Li et al., 1988; Royds & Iacopetta, 2006). Importance is given to genetic counselling preventive measures in LFS patients and their families, where such individuals are closely monitored, advised on ways to avoid DNA damaging agents, as well as preventative mammograms or mastectomies offered to the female members of the families (Royds & Iacopetta, 2006).

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Neurological disorders

Other than in neoplastic diseases, p53 plays a role in the progression of neurodegenerative diseases such as Parkinson’s disease (PD) as a response to cellular stress, resulting in the damage, degradation, and death of neurons (Jõers et al., 2004). Such neurodegenerative diseases are characterised by the accumulation of abnormal protein aggregates and damaged mitochondria (Chu, 2019 ; Matoba et al., 2006). For example in PD, high levels of p53 leads to the damage of mitochondria and production of reactive oxygen species within the cellular environment which in turn causes further cellular oxidative stress (Luo et al., 2022). Impaired autophagy has also been suggested to contribute to the accumulation of proteins, where cytosolic p53 decreases the ubiquitin-mediated degradation of α-synuclein protein, accumulating at the presynaptic terminal and leading to neurodegeneration in PD (Luo et al., 2022). Animal and cellular models have shown that the pharmacological inhibition of p53 is effective against the loss of dopaminergic neurons (Luo et al., 2022; Royds & Iacopetta, 2006).

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Atherosclerosis and Coronary Heart Disease

Atherosclerosis is the progressive thickening and hardening of the arteries due to the accumulation of plaque composed of lipid and fibrous elements within the vessel walls, leading to coronary heart disease and stroke, with an incidence as high as 50% in the western world (Lusis, 2000). P53 has also been linked to disease progression due to the impact p53 has on proliferation and apoptosis. A study involving apoE-knockout mice models, representing families suffering from  hypercholesterolemia, with p53 inactivation have shown that dysfunction of p53 leads to increased atherogenesis and a significantly accelerated rate of cellular proliferation when compared to those mice with functional p53 (Chan et al., 1999). Furthermore, this study also linked the absence of p53 to an increase in macrophage proliferation towards the fatty core of the plaques, as well as increased necrosis and reduced collagen deposits, leading to the production of dangerous and unstable plaque formation (Chan et al., 1999).

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Conclusion

In conclusion, the journey of understanding p53 began with its discovery in association with the SV40 virus, initially mischaracterized as a proto-oncogene. Subsequent investigations, notably by Eliyahu et al. (1984), suggested an oncogenic role; yet, thorough studies by Finlay et al. (1989) corrected this misconception, firmly establishing p53 as a tumour suppressor. Expanding on p53's structure, its transactivation domain plays a pivotal role in target gene transcription, interacting with the transcription machinery. The proline-rich region contributes to growth suppression, while the core DNA-binding domain ensures sequence-specific binding. Tetramerization, regulated by distinct domains, influences subcellular localization and DNA binding. Recent discoveries of p53 isoforms underscore its complexity, offering potential insights into cancer biology and prognosis. Furthermore, the DNA damage response mediated by p53 is a pivotal safeguard mechanism in cellular homeostasis. Initially identified by Kastan et al. in 1991, p53's role in coordinating cell cycle arrest and DNA repair following DNA damage underscores its significance in maintaining genomic integrity. Aberrations in p53 function, attributed to mutations or dysregulation, contribute to various diseases, notably cancer, exemplifying its dual nature as a tumor suppressor and, under certain conditions, an oncogene. Furthermore, p53's involvement in autophagy, apoptosis, and metabolic regulation emphasises its multifaceted impact on cellular processes. Understanding these intricate pathways unveils potential therapeutic strategies, offering hope in the quest for effective treatments against cancer and other p53-associated disorders.

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