Proctor and Gray (2008), The p53-Mdm2 System
The p53 protein is a sequence-specific DNA binding transcription factor which is encoded by TP53 gene (in human) located on the short arm of chromosome 17. The ability of p53 to induce cell growth arrest and apoptosis is relatively well-understood, and its importance in tumour suppression - p53 was identified as a tumour suppressor gene - is firmly established. An article by Jin and Levine (2001)  explains the functional circuit of p53.
The cellular concentration of p53 must be tightly regulated. While it can suppress tumours, a high level of p53 may accelerate the ageing process by excessive apoptosis. In normal (unstressed) cells, p53 protein levels are low because of a continuous degradation of p53. This is done by the Mdm2 protein (also called HDM2 protein), an ubiquitin E3 ligase, upon binding to the N-terminal transactivation domain (TAD) of p53. p53 also acts as a transcriptional activator, regulating the expression of Mdm2 (for its own regulation). Mdm2 binding on p53 prevents its activity and targets it from the nucleus to the proteasome in the cytosol for degradation.
The p53 protein becomes activated in response to DNA damage (induced by either UV, IR or chemical agents, such as hydrogen peroxide) and other stress signals such as oxidative stress, osmotic shock, ribonucleotide depletion and deregulated oncogene expression. Following this, the cell is required to arrest its cell cycle to allow DNA repair to take place, in order to prevent replication of damaged DNA. p53 has three major functions: growth arrest, DNA repair and apoptosis (cell death). The major activities of p53 and Mdm2 proteins are illustrated in figure 1 (taken from ).
Figure 1: Major activities implicating p53 and Mdm2. Figure taken from .
DNA damage is sensed by 'checkpoints' in the cell cycle, and causes protein kinases such as ATM, ATR, CHK1 and CHK2 to phosphorylate p53 at the N-terminal transcriptional activation domain which has a large number of phosphorylation sites (sites that are close to or within the MDM2-binding site of p53). Phosphorylation of the N-terminal end of p53 by kinases disrupts Mdm2-binding. In addition, Mdm2 also gets phosphorylated which also enhances the degradation of Mdm2. The activation of p53 is also stimulated by oncogenes, mediated by the nucleolar protein ARF (p14ARF in humans) that senses DNA damage. ARF directly interacts with Mdm2, and leads to up-regulation of p53 transcriptional response. ARF sequesters Mdm2 in the nucleolus, resulting in inhibition of nuclear export and activation of p53, since nuclear export is essential for proper p53 degradation.
The activation of p53 is marked by two major events. First, the half-life of the p53 protein is increased drastically, leading to quick accumulation of p53 in stressed cells. Secondly, a conformational change induced by proteins such as Pin1 forces p53 to take on an active role as a transcription regulator in these cells. Transcriptional coactivators, like p300 or PCAF, then acetylate the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes. The regulation and major activities implicated for p53 are illustrated in figure 2 (taken from ).
Figure 2: DNA damage induces phosphorylation (P) of p53 (at the Mdm2 binding site) and Mdm2, preventing Mdm2 from binding to p53. As a result, the p53 level increases, and stops cells from entering cell cycle until the DNA is repaired. If repair fails, p53 initiates apoptosis (programmed cell death). Mechanisms resulting in a decrease in p53 steady-state levels are indicated with green arrows and those resulting in increases p53 levels are indicated with red arrows. Solid lines indicate active mechanisms and broken lines indicate inactivated mechanisms. Figure taken from .
In the cell, p53 protein binds DNA, which in turn stimulates another gene to produce a protein called p21 that interacts with a cell division-stimulating protein (cdk2). When p21 is complexed with cdk2, the cell cannot pass through to the next stage of cell division. The growth arrest stops the progression of cell cycle, preventing replication of damaged DNA. Mutant p53 can not longer bind DNA in an effective way, and as a consequence the p21 protein is not made available to act as the "stop signal" for cell division. Thus cells divide uncontrollably, and form tumours .
During growth arrest, p53 may activate the transcription of proteins involved in DNA repair. One of its transcriptional target genes, p53R2, encodes ribonucleotide reductase, which is important for both DNA replication and repair. p53 also interacts directly with AP endonuclease and DNA polymerase, which are involved in base excision repair.
Apoptosis is the "last resort" to avoid proliferation of cells containing abnormal DNA. Apoptosis can be induced by the binding of Caspase 9 to cytochrome c and Apaf1. p53 may activate the expression of Apaf1 and Bax. The latter can then stimulate the release of cytochrome c from mitochondria.
In the article presented here , the authors have developed two independent models (models of p53 stabilisation through 1) ARF (BIOMD0000000189) and 2) ATM (BIOMD0000000188), respectively) to see whether oscillations of p53 and Mdm2 would result from either of these mechanisms. The authors have described two stochastic mechanistic models of the p53/Mdm2 circuit and show that sustained oscillations result directly from the key biological features, without assuming complicated mathematical functions or requiring more that one feedback loop. Each model examines a different mechanism for providing a negative feedback loop which results in p53 activation after DNA damage. This first model (ARF model) looks at the mechanism of p14ARF which sequesters Mdm2 and leads to stabilisation of p53. The second model (ATM model) examines the mechanism of ATM activation which leads to phosphorylation of both p53 and Mdm2 and increased degradation of Mdm2, which again results in p53 stabilisation.
Stochastic simulation was used since this is the most natural way to introduce the cellular variability which is seen experimentally. figure 3 (panel A and B) shows the stochastic simulation result for ARF and ATM models. Oscillations are triggered when DNA damage accumulates gradually over time and disappear when DNA is repaired (ARF model) (figure 4). In order to show that the variability in the oscillations is qualitatively different from those seen in deterministic simulations, the authors have also performed deterministic simulations on the models (figure 3, panel C and D). In the ARF model, oscillations are still produced but interestingly, the deterministic version of the ATM model predicts only one peak followed by fairly constant levels of total p53 and Mdm2 but at a level higher that the initial values. The deterministic model loses the oscillations due to averaging effects. The averaging effect is due to inter-cell variability in the oscillatory period and although the cells are synchronised for the first peak, they are unsynchronised for all the following peaks. Therefore the oscillations in the different cells cancel out. This is also observed experimentally if measurements are taken for a population of cells rather than individual cells.
Figure 3: Simulation results for ARF and ATM models. Panels A and C show stochastic and deterministic simulation plots for the ARF model and panels B and D show the stochastic and deterministic simulation results for the ATM model under conditions of irradiation (IR=25 dGy for 1 minute at time t = 1 hour. p53 is shown in green, Mdm2 in red. Figure assembled from various figures in .
Figure 4: Oscillations are triggered when DNA damage accumulates gradually over time and disappears when DNA is repaired (ARF model). p53 is shown in green, Mdm2 in red. Figure taken from .
Cellular mechanisms that contribute to the variability in the pattern of sustained oscillations after DNA damage are examined by the ARF and ATM model of the p53-Mdm2 circuit. The models predict more regular oscillations if ARF is present and suggest the need for further experiments in ARF positive cells to test these predictions. The work illustrates the importance of systems biology approaches to understanding the complex role of p53 in both ageing and cancer.
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- National Center for Biotechnology Information. The p53 tumor suppressor protein in: Genes and Disease, 2009.
- C.J. Proctor and D.A. Gray. Explaining oscillations and variability in the p53-Mdm2 system. BMC Syst Biol 2:75, 2008. [SRS@EBI]