Dynamics in the p53-Mdm2 Ubiquitination Pathway

The tumor suppressor p53 is highly regulated under various states of cellular stress. p53 stability is predominantly regulated through the ubiquitin-proteasomal pathway by the E3 ligase Mdm2. p53 ubiquitination is a dynamic process with Mdm2 capable of catalyzing both mono- and polyubiquitination. Additionally, deubiquitination is an important step occurring in p53 and Mdm2 stabilities. Factors such as HAUSP, p14ARF, and MdmX play important regulatory roles in p53 ubiquitination/deubiquitination and their interplay with Mdm2 and p53 compound layers of complexity for regulating this important pathway.     

p53-Mdm2模块和泛素化系统

p53(肿瘤抑制基因)诱导鼠双微粒体蛋白2(Mdm2)的表达,Mdm2反之抑制p53的活性,Mdm2和p53形成了一个自动调整的模块。Mdm2的一个重要的结构标志是一个中心酸性区域,另外的结构标志是在酸结构域下游的一个锌指结构,和一个C端的环指区域。Mdm2的表达是由p53来调节,Mdm2作为E3连接酶使p53泛素化并且驱使p53降解,进而控制p53的功能。对于p53泛素化的结构要求是p53的寡聚化。p53泛素化作用的调整模式是通过蛋白质之间的相互作用。Mdm2中环指区域的作用是通过使p53泛素化来推进p53的降解。泛素化后的酸性结构在Mdm2的降解中起作用。     

Bifurcation in Cell Cycle Dynamics Regulated by p53

We study the regulating mechanism of p53 on the properties of cell cycle dynamics in the light of the proposed model of interacting p53 and cell cycle networks via p53. Irradiation (IR) introduce to p53 compel p53 dynamics to suffer different phases, namely oscillating and oscillation death (stabilized) phases. The IR induced p53 dynamics undergo collapse of oscillation with collapse time Δt which depends on IR strength. The stress p53 via IR drive cell cycle molecular species MPF and cyclin dynamics to different states, namely, oscillation death, oscillations of periods, chaotic and sustain oscillation in their bifurcation diagram. We predict that there could be a critical Δt _c induced by p53 via IR _c, where, if Δt〈Δt _c the cell cycle may come back to normal state, otherwise it will go to cell cycle arrest (apoptosis).     

The RP-p53-Mdm2 pathway

Comment on: Llanos S, et al. Cell Cycle 2010; 9:4005–12.     

Skp2 suppresses p53-dependent apoptosis by inhibiting p300

The F box protein Skp2 is oncogenic, and its frequent amplification and overexpression correlate with the grade of malignancy of certain tumors. Conversely, depletion of Skp2 decreases cell growth and increases apoptosis. Here, we show that Skp2 counteracts the transactivation function of p53 and suppresses apoptosis mediated by DNA damage or p53 stabilization. We demonstrate that Skp2 forms a complex with p300 through the CH1 and the CH3 domains of p300 to which p53 is thought to bind and antagonizes the interaction between p300 and p53 in cells and in vitro. As Skp2 antagonizes the interaction between p300 and p53, Skp2 suppresses p300-mediated acetylation of p53 and the transactivation ability of p53. Conversely, ectopic expression of p300 rescues the transactivation function of p53 in cells overexpressing Skp2. Taken together, our results indicate that Skp2 controls p300-p53 signaling pathways in cancer cells, making Skp2 a potential molecular target for cancer therapy.     

A theoretical exploration of birhythmicity in the p53-Mdm2 network

Experimental observations performed in the p53-Mdm2 network, one of the key protein modules involved in the control of proliferation of abnormal cells in mammals, revealed the existence of two frequencies of oscillations of p53 and Mdm2 in irradiated cells depending on the irradiation dose. These observations raised the question of the existence of birhythmicity, i.e. the coexistence of two oscillatory regimes for the same external conditions, in the p53-Mdm2 network which would be at the origin of these two distinct frequencies. A theoretical answer has been recently suggested by Ouattara, Abou-Jaoudé and Kaufman who proposed a 3-dimensional differential model showing birhythmicity to reproduce the two frequencies experimentally observed. The aim of this work is to analyze the mechanisms at the origin of the birhythmic behavior through a theoretical analysis of this differential model. To do so, we reduced this model, in a first step, into a 3-dimensional piecewise linear differential model where the Hill functions have been approximated by step functions, and, in a second step, into a 2-dimensional piecewise linear differential model by setting one autonomous variable as a constant in each domain of the phase space. We find that two features related to the phase space structure of the system are at the origin of the birhythmic behavior: the existence of two embedded cycles in the transition graph of the reduced models; the presence of a bypass in the orbit of the large amplitude oscillatory regime of low frequency. Based on this analysis, an experimental strategy is proposed to test the existence of birhythmicity in the p53-Mdm2 network. From a methodological point of view, this approach greatly facilitates the computational analysis of complex oscillatory behavior and could represent a valuable tool to explore mathematical models of biological rhythms showing sufficiently steep nonlinearities.     

Cyclin G: a regulator of the p53-Mdm2 network

A recent study published in the April issue of Molecular Cell has shown that cyclin G, a p53 target, is a regulatory component of the active PP2A holoenzyme, which activates Mdm2 through dephosphorylation. These findings suggest that cyclin G is a key regulator of the p53-Mdm2 network.     

A dynamic role of HAUSP in the p53-Mdm2 pathway

Our previous study showed that ubiquitination of p53 is reversible and that the ubiquitin hydrolase HAUSP can stabilize p53 by deubiquitination. Here, we found that partial reduction of endogenous HAUSP levels by RNAi indeed destabilizes endogenous p53; surprisingly, however, nearly complete ablation of HAUSP stabilizes and activates p53. We further show that this phenomenon occurs because HAUSP stabilizes Mdm2 in a p53-independent manner, providing an interesting feedback loop in p53 regulation. Notably, HAUSP is required for Mdm2 stability in normal cells; in HAUSP-ablated cells, self-ubiquitinated-Mdm2 becomes extremely unstable, leading to indirect p53 activation. Furthermore, this feedback regulation is specific to Mdm2; in HeLa cells, where p53 is preferentially degraded by viral E6-dependent ubiquitination, depletion of HAUSP fails to activate p53. This study provides an example of an ubiquitin ligase (Mdm2) that is directly regulated by a deubiquitinase (HAUSP) and also reveals a dynamic role of HAUSP in the p53-Mdm2 pathway.     

The Pharmacodynamics of the p53-Mdm2 Targeting Drug Nutlin: The Role of Gene-Switching Noise

In this work we investigate, by means of a computational stochastic model, how tumor cells with wild-type p53 gene respond to the drug Nutlin, an agent that interferes with the Mdm2-mediated p53 regulation. In particular, we show how the stochastic gene-switching controlled by p53 can explain experimental dose-response curves, i.e., the observed inter-cell variability of the cell viability under Nutlin action. The proposed model describes in some detail the regulation network of p53, including the negative feedback loop mediated by Mdm2 and the positive loop mediated by PTEN, as well as the reversible inhibition of Mdm2 caused by Nutlin binding. The fate of the individual cell is assumed to be decided by the rising of nuclear-phosphorylated p53 over a certain threshold. We also performed in silico experiments to evaluate the dose-response curve after a single drug dose delivered in mice, or after its fractionated administration. Our results suggest that dose-splitting may be ineffective at low doses and effective at high doses. This complex behavior can be due to the interplay among the existence of a threshold on the p53 level for its cell activity, the nonlinearity of the relationship between the bolus dose and the peak of active p53, and the relatively fast elimination of the drug.     

A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer cell growth and survival and has been implicated in cancer pathophysiology and treatment. Here we report a novel function for IGF-1R in p53-dependent apoptotic response. We show that inhibition or loss of IGF-1R activity reduces translational synthesis of p53 and Mdm2 protein. Notably, IGF-1R inhibition increases p53 protein stability by reducing p53 ubiquitination and maintains p53 at low levels by decreasing p53 synthesis, thus rendering p53 insensitive to stabilization after DNA damage. The accumulation and apoptosis of DNA-damage-induced p53 is therefore reduced in Igf-1r(-/-) mouse embryonic fibroblasts or tumor cells treated with the IGF-1R inhibitor. Furthermore, we find that inhibition of IGF-1R reduces p53 and Mdm2 translation through a gene-specific mechanism mediated by the respective 5' untranslated region of p53 and mdm2 messenger RNA. The eukaryotic translation initiation factor 4F complex is also involved in this translational inhibition. These results demonstrate an unexpected role for translational control by IGF-1R in p53-mediated apoptosis.     

p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.     

Benzimidazole-2-one: A novel anchoring principle for antagonizing p53-Mdm2

Herein we propose the benzimidazole-2-one substructure as a suitable tryptophan mimic and thus a reasonable starting point for the design of p53 Mdm2 antagonists. We devise a short multicomponent reaction route to hitherto unknown 2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamides by reacting mono N-carbamate protected phenylenediamine in a Ugi-3CR followed by base induced cyclisation. Our preliminary synthesis and screening results are presented here. The finding of the benzimidazolone moiety as a tryptophan replacement in mdm2 is significant as it offers access to novel scaffolds with potentially higher selectivity and potency and improved biological activities. Observing low μM affinities to mdm2 by NMR and fluorescence polarization we conclude that the 2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamide scaffold might be a good starting point to further optimize the affinities to Mdm2.     

In silico criterion for prediction of effects of p53 gene missense mutations on p53-Mdm2 feedback loop

The Informational Spectrum Method (ISM) is the tool for the in silico analysis of proteins which interprets protein sequence linear information using signal analyses methods. In this paper the ISM was employed to characterize the products of genetic variants of tumor suppressor gene p53 and its natural binding regulator protein Mdm2. Based on this we propose the criterion for identification of missense mutations that have impact on the p53-Mdm2 feedback loop. The efficiency of the proposed criterion was confirmed by the ISM analyses of p53 mutants reported in: (i) healthy individuals, (ii) germline mutations database and (iii) somatic mutations database.