Regulation and function of celebrity molecule p53

P53 is the most important tumor suppressor gene, with broad and powerful functions. Since the discovery of p53 protein in 1979, p53 has been a "star molecule" in molecular biology and oncology. Using p53 as a keyword search in the PubMed database, over 100000 articles can be found.

1. Regulation of p53: Post translational modifications of proteins are key

In order to accurately perform its multifaceted functions, the expression and activity of p53 require precise and multi-level regulation at the protein, DNA, and RNA levels.

2. Regulation at the protein level

The p53 protein can undergo various types of PTMs, including ubiquitination, phosphorylation, acetylation, sumylation, etc. Different stimulation signals determine the site and type of PTMs.

Many PTMs of p53 are reversible. The effects of PTM on p53 include altering its protein levels, cellular localization, cofactor recruitment, target gene selectivity, and even protein aggregation. Among them, ubiquitination, phosphorylation, and acetylation are the most common and influential in affecting p53 function.

1) Ubiquitination

Ubiquitination usually occurs at the C-lysine residue of p53. MDM2 is the most famous p53 regulatory factor, which causes ubiquitination and degradation of p53 to maintain low p53 levels in unstimulated cells or export nuclear p53 to the cytoplasm. Many stimuli and regulatory factors activate p53 by reducing the inhibition of MDM2. For example, p14ARF protein can stabilize p53 by inhibiting MDM2 mediated degradation of p53. The convincing evidence supporting the importance of MDM2 mediated p53 inhibition comes from mouse models, where MDM2 deficiency induced embryonic lethality can be restored by knocking out p53.

Interestingly, MDM2 itself is a target gene of p53. The feedback loop formed by MDM2-p53 is the core of p53 related pathways. MDMX (or MDM4) is a member of the MDM2 family, and although it lacks E3 ubiquitin ligase activity, it can form heterodimers with MDM2, enhancing the degradation of p53. Other E3 ubiquitin ligases that can degrade p53 include ARF-BP1/HUWE1, COP1, CHIP, and Pirh2.

2) Phosphorylation

The phosphorylation sites of serine and threonine are distributed throughout the p53 protein. The phosphorylation of p53 by ATM was the earliest mechanism used to explain how p53 responds to DNA damage. The phosphorylation of S15, T18, and S20 disrupts the binding and inhibition of MDM2 to p53, while enhancing the interaction between p53 and transcription factors such as CBP. The result is the activation of p53 mediated transcription, inducing cell cycle arrest and apoptosis. Severe DNA damage further phosphorylates p53 at S46, thereby enhancing cell apoptosis.

3) Acetylation

The discovery of p53 acetylation is the earliest example of non histone acetylation. Acetylation of several lysine residues in DBD is critical to the ability of p53 activation, which can affect cell cycle arrest, apoptosis, aging, iron death and mTOR pathway in a promoter specific manner. The role of p53 acetylation in tumor suppression can be best demonstrated through a series of acetylation deficient (KR mutation) gene knock in mouse models. For example, although the p53-3KR mutant retains its DNA binding activity, it cannot activate major target genes such as p21. However, p53-3KR mutant mice are less susceptible to tumors. The p53-4KR and p53-5KR mutants further eliminated the ability of p53 to regulate ferroptosis and mTOR pathway, resulting in the basic loss of tumor suppressive function of p53.

It is worth noting that the acetylation of CTD is complex: lysine residues identical to CTD can also undergo methylation, ubiquitination, SUMOylation, and NEDDylation modifications. Therefore, CTD acetylation deficient mutants (p53-6KR and p53-7KR) mice did not show significant effects on tumor suppression, as these mutants simultaneously eliminated the positive and negative effects of different types of PTM on p53 function. In fact, mutant mice mimicking acetylation (p53 KQ) showed significant p53 activation in transcription and tumor suppression (even though p53 protein levels did not significantly increase), highlighting the role of CTD acetylation in the in vivo state.

In summary, a variety of protein modification types together regulate the activity of p53. It is worth noting that although in vitro studies have shown that many PTMs have important effects on the function of p53, these results may not be reproducible in vivo. The most suitable way to understand the effect of a certain protein modification on the function of p53 is to use a knock in mouse model.

At the protein level, cofactor is another important factor affecting p53 activity. The p53 protein can bind to various cofactors, including activators and inhibitors, which have important effects on its protein folding, stability, cell localization, DNA binding, transcriptional activation ability, and target gene selection. For example, many molecular chaperones can regulate the folding and stability of p53. MDM2 and MDMX bind to the TAD of p53 to inhibit its transcriptional activation activity (independent of the role of MDM2 as an E3 ubiquitin ligase). Multiple transcription regulatory factors, such as PBRM1, SET, and Dicer, can interact with p53. The components of m6A methyltransferase complex, METTL3 and RBM15, interact with p53 and selectively modify the mRNA of some p53 target genes.

3. Regulation at the DNA and RNA levels

The p53 gene has two promoters, leading to selective transcription initiation. The promoter region of the p53 gene can undergo DNA methylation and histone methylation, thereby affecting the transcription of the p53 gene itself. Multiple transcription factors can control the transcription of p53 gene. The pre mRNA of p53 can undergo alternative splicing. In addition, the stability, cellular localization, and translation of p53 mRNA are strictly regulated. In addition, the activation of p53 is not a simple "all or nothing" pattern, but a highly dynamic process. The heterogeneity of cells, the characteristics of stimuli, the diverse regulatory factors mentioned above, and the stability of downstream target genes collectively determine the dynamic changes in p53 activity.

4. The function of p53: diversity and complexity

P53 can regulate multiple functions, which together form a complex p53 functional network.

The function of p53 and its role in physiological and pathological processes

5. Cell cycle arrest, apoptosis, aging, and genomic stability

Inducing cell cycle arrest, apoptosis, and aging are the earliest discovered functions of p53. Various stimulation signals can induce p53 to exert these functions, among which DNA damage is the most effective type of stimulation. After DNA damage, p53 protein is stabilized and activated to prevent cell cycle progression, providing cells with a time window and sufficient material and energy to repair damaged DNA. If the damage is too severe to be repaired, p53 will trigger cell apoptosis and aging to eliminate the damaged cells. It is worth noting that the outcome of p53 activation also depends on the type of cell and DNA damage. These three functions are widely regarded as the main barriers for p53 to prevent tumor development. In addition, p53 can also be activated by DNA damage generated during CRISPR-Cas9 genome editing, thereby reducing the efficiency of gene editing.

On the other hand, failure to eliminate damaged cells can lead to genomic instability. The loss of p53, including loss of heterozygosity (LOH) and inactivation or deletion of alleles, can promote genomic instability and drive the evolution of tumor cell genomes. P53 is known as the 'guardian of the genome'. P53 can directly promote DNA damage repair. In fact, many p53 target genes contribute to the DNA repair process. However, it is still unclear whether the activation of these DNA repair related target genes mediated by p53 is sufficient to inhibit tumor development independently of other p53 functions.

6. Metabolism and iron death

P53 is an important regulatory factor that regulates glucose, lipids, amino acids, nucleotides, iron, and redox metabolism. In addition, it also regulates autophagy and has extensive interactions with key metabolic regulatory factors such as AMPK, AKT, and mTOR. The function of p53 is associated with various metabolic diseases, including cancer. Generally speaking, p53 inhibits synthetic metabolism processes (such as de novo lipogenesis and nucleotide synthesis) while promoting catabolism (including oxidative phosphorylation, lipolysis, and fatty acid oxidation). Enhanced glycolysis produces various molecular materials for cancer cell biosynthesis (Warburg effect), which is also inhibited by p53. These metabolic functions of p53 inhibit the need for rapid proliferation of cancer cells, leading to tumor suppression. However, p53 exhibits bidirectional effects in many metabolic processes. The essence of this contradictory activity lies in the complexity of p53 function. The role of p53 in ROS control provides a good example. When ROS intensity is low (representing mild, transient, tolerable, and modifiable stimuli), p53 exerts antioxidant effects, reduces ROS, and protects cells from damage (promoting survival). On the contrary, when ROS levels are too high and may cause uncontrollable damage (representing severe, long-term, harmful, and irretrievable stimuli), p53 further increases ROS levels, leading to cell death and protecting nearby undamaged cells (promoting death).

Iron dependent regulated cell death occurs when lipid peroxidation levels are too high and is closely related to metabolic pathways. P53 is one of the main regulatory factors of ferroptosis. Several metabolic target genes of p53, including SLC7A11, VKORC1L1, GLS2, and PLTP, are directly involved in regulating ferroptosis. Similar to apoptosis during DNA damage response, ferroptosis can eliminate severely damaged cells during metabolic stress. P53 mediated ferroptosis is considered an important weapon in tumor suppression. It is interesting that an African population specific p53 SNP may weaken the ability of p53 to induce ferroptosis, thereby impairing its tumor suppressive function. Unlike apoptosis, the occurrence of classical ferroptosis typically requires treatment of cells with ferroptosis inducers (such as GPX4 inhibitors). It is worth noting that a recent study has shown that in the absence of common iron death inducers, PHLDA2 mediated phosphatidic acid peroxidation triggers non classical iron death reactions. An interesting question is which iron death pathway plays a more important role in p53 mediated tumor suppression, as p53 can promote both classical and non classical iron death processes simultaneously.

7. Stem cells compete with cells

Stem cells have many similarities with cancer cells, including sustained proliferation ability, metabolic reprogramming, and a core transcription factor network. Therefore, p53 restricts cell stemness and regulates cell fate in various types of stem cells. In embryonic stem cells (ESCs), p53 inhibits genes that maintain stemness while activating genes that promote differentiation. In adult stem cells (ASCs), p53 inhibits cell self-renewal, promotes stem cell exhaustion, maintains stem cell homeostasis, or stimulates differentiation. The ability of p53 to restrict cell stemness is crucial for its tumor suppressive function, as it can hinder the formation of cancer stem cells. The specific differentiation pathway regulated by p53 contributes to tumor suppression in lung cancer. The loss or mutation of p53 may lead to dedifferentiation, cell reprogramming, and increased cell plasticity in cancer. The process of generating induced pluripotent stem cells (iPSCs) is similar to dedifferentiation and cellular carcinogenesis. P53 is one of the main inhibitory factors in this process, and silencing p53 can significantly improve the efficiency of iPSC production. Cellular competition is crucial in normal development, tissue damage repair, tumor evolution, and metastasis.

Generally speaking, due to the inhibition of synthesis metabolism and proliferation by p53, while promoting cell death, it is not conducive to cells outperforming neighboring cells in competition. Therefore, high levels of p53 activity often indicate a "loser" state in cell competition. However, studies have shown that the super competitive cells in fruit flies require p53 activity to eliminate nearby normal cells. The regulation of cell competition by p53 has important physiological significance. Cells carrying mutant p53 may undergo clonal expansion, which may drive the occurrence and evolution of tumors. These p53 mutant cells are not always preserved, as they may undergo necrotic apoptosis by competing with nearby normal cells, or be eliminated by cells with other gene mutations that have higher fitness.

8. Tumor metastasis

P53 inhibits metastasis at multiple stages in both autonomous and non autonomous ways. In tumor cells, p53 restricts their mobility and epithelial mesenchymal transition (EMT) process. Metastatic cancer cells in the circulatory system may undergo anoikis and ferroptosis, both of which can be promoted by p53 to prevent cancer cells from migrating to new sites. At every step of metastasis and diffusion, cancer cells adopt specialized metabolic programs to meet their energy and biomolecule needs, and p53 may inhibit these cellular metabolic processes. On the other hand, p53 shapes a tumor microenvironment (TME) that is unfavorable for metastasis. For example, p53 inhibits angiogenesis and lymphangiogenesis, blocking the pathways of metastasis through the blood and lymphatic systems. It can also maintain the integrity of the extracellular matrix, enhance the adhesion of tumor cells on it, and restrict the movement of tumor cells. In addition, p53 can also activate and inhibit the inflammatory response of tumor metastasis.

9. Immunity

Another important function of p53 is to regulate immune responses. P53 plays a role in innate and adaptive immunity through multiple mechanisms. The synergistic construction of tumor suppressive immune network by p53 in tumor cells and non tumor cells. In tumor cells, p53 indirectly inhibits PD-L1 expression by upregulating miR-34, making tumor cells sensitive to anti-tumor immune response and immunotherapy. P53 activates the cGAS STING pathway to induce anti-tumor activity. In a mouse liver cancer model, restoring p53 expression induces tumor cell senescence, triggers the release of inflammatory cytokines, and triggers innate immune responses to eliminate tumor cells.

In hepatic stellate cells, p53 induced cellular senescence also exhibits liver cancer inhibitory effects - by establishing senescence associated secretory phenotype (SASP), M1 macrophage polarization can be enhanced to maintain tumor suppressive TME. In subtypes of mouse bone marrow precursor cells, p53 drives their differentiation into Ly6c+CD103+monocytic antigen-presenting cells, thereby enhancing anti-tumor immunity.

The loss of p53 in tumor cells or TME cells can reverse the tumor suppressive immune microenvironment to an immunosuppressive state, promote immune tolerance or escape of tumor cells, or establish an inflammatory environment conducive to tumor metastasis. Mutant p53 may stimulate tumor cell immune evasion. Interestingly, p53 mutants themselves can generate tumor antigens as targets for tumor immunotherapy.

P53 also participates in autoimmune reactions and immune defense against various pathogens. It is worth noting that not all immune related activities of p53 promote immune cell function or are beneficial to health. P53 may inhibit the proliferation and function of certain T cell subtypes. For example, p53 inhibits antigen-specific CD4+T cell proliferation, which can be eliminated through the T cell receptor (TCR) signaling pathway. Some viruses rely on p53 to cause cell cycle arrest for replication.