Mathematical modelling of the core regulatory feedback mechanisms of p53 protein that decide cell fate

Abstract

Cells defence against stresses that can cause DNA damage (single-strand breaks, double-strand breaks) is crucial in safeguarding the integrity of the genome and the survival of the organism as a whole. One of the genes that plays a pivotal role in maintaining the stability of the genome in humans is p53, which encodes its product p53 protein. The regulation of p53 activation is extremely complex, and molecular cell biology has gathered parts and pieces of the whole pathway. Mental intuition of this complex regulation is challenging; therefore, it requires a different method to quantitatively model and analyse to enhance the current understanding. This thesis has attempted to create two quantitative models of the mechanisms that regulate p53 basal levels and its appropriate activation as a stress response in deciding cell fate by either cell cycle arrest (to stop proliferation of DNA-damaged cells) or apoptosis (programmed cell death) to eliminate damaged cells. In the first part of the research, a modified and improved model from Sun et al. (2011) deterministic model is proposed to explain the p53 basal dynamics and its response to stress due to DNA double-strand breaks. This model in the form of delay differential equations incorporates the most recently found molecular interactions and hypothesis: the core regulators consist of ATM, Mdm2, MdmX, Wip1 and p53. ATM as a stress transducer, amplifies the stress signal and activates p53 and inhibits its regulators Mdm2 and MdmX. The network structure consists of two positive feedback loops (p53 auto-regulation and ATM auto-activation), three negative feedback loops (Mdm2, MdmX and Wip1) and the interplay of p53, Mdm2 and MdmX that have successfully captured the basal dynamics (spontaneous pulses under non-stressed conditions) and stress response (repeated pulses or oscillations under stressed conditions). The model simulation results show that p53 spontaneous pulses are due to intrinsic DNA damage involving low number of DNA double-strand breaks; and p53 auto-regulation is an important positive feedback contributing to a threshold activation of p53 in generating pulses whether spontaneous or repeated. It also shows that p53 dynamics are excitable, in that once initiated, it completes the pulse even if stress signal is inhibited. Bifurcation analysis revealed a spectrum of p53 behaviour under stressed and non-stressed conditions and characterised p53 dynamics as Type II excitability (oscillations arises from non-zero frequency). Most importantly, we reveal some novel findings on the mechanism of threshold activation of p53 pulsatile and oscillatory dynamics that are crucial for its physiological function as a transcription factor and guardian of the genome. The second model is an extension of the first model by incorporating the apoptosis initiation module structure from Zhang et al. (2009a) with modified parameter values for modelling the core regulatory mechanism of p53 protein that activates apoptotic switch in response to high DNA double-strand breaks. The apoptosis initiation module includes Puma, Bcl2 and Bax. p53 activates the transcription of Puma (BH3-only protein that is pro-apoptotic) as a trigger of apoptosis that inhibits Bcl2 protein (pro-survival) and directly activates Bax. Activation of Bax was assumed to be an indicator of apoptosis initiation. The constructed model demonstrated how molecular interactions and stress signal amplification from ATM auto-activation in the p53 network control cell life and death decisions. Particularly, the model simulation results are qualitatively consistent with the experimental findings of an all-or-none activation of apoptosis and predicted overexpression of Bcl2 as a factor in causing the malfunction of the apoptotic switch. This model presents a simplified yet plausible model for molecular mechanism that regulates p53 activation of the apoptotic switch. The model gives insight into the design principles underlying p53 regulation of apoptosis. In summary, the two models presented in this thesis have proposed plausible design principles of p53 basal dynamics and DNA damage response, and activation of apoptotic switch. These models provide novel theoretical insights into p53 regulation.