Frequency domain analysis reveals external periodic fluctuations can generate sustained p53 oscillation

p53 is a well-known tumor suppressor protein that regulates many pathways, such as ones involved in cell cycle and apoptosis. The p53 levels are known to oscillate without damping after DNA damage, which has been a focus of many recent studies. A negative feedback loop involving p53 and MDM2 has been reported to be responsible for this oscillatory behavior, but questions remain as how the dynamics of this loop alter in order to initiate and maintain the sustained or undamped p53 oscillation. Our frequency domain analysis suggests that the sustained p53 oscillation is not completely dictated by the negative feedback loop; instead, it is likely to be also modulated by periodic DNA repair-related fluctuations that are triggered by DNA damage. According to our analysis, the p53-MDM2 feedback mechanism exhibits adaptability in different cellular contexts. It normally filters noise and fluctuations exerted on p53, but upon DNA damage, it stops performing the filtering function so that DNA repair-related oscillatory signals can modulate the p53 oscillation. Furthermore, it is shown that the p53-MDM2 feedback loop increases its damping ratio allowing p53 to oscillate at a frequency more synchronized with the other cellular efforts to repair the damaged DNA, while suppressing its inherent oscillation-generating capability. Our analysis suggests that the overexpression of MDM2, observed in many types of cancer, can disrupt the operation of this adaptive mechanism by making it less responsive to the modulating signals after DNA damage occurs.     

Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFκB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFκB peptide (residues 303–332) from its complex with ASPP2 Ank-SH3, indicating that NFκB 303–332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFκB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFκB via distinct sites.     

Regulation of the HDM2-p53 pathway by ribosomal protein L6 in response to ribosomal stress

The HDM2-p53 loop is crucial for monitoring p53 level and human pathologies. Therefore, identification of novel molecules involved in this regulatory loop is necessary for understanding the dynamic regulation of p53 and treatment of human diseases. Here, we characterized that the ribosomal protein L6 binds to and suppresses the E3 ubiquitin ligase activity of HDM2, and subsequently attenuates HDM2-mediated p53 polyubiquitination and degradation. The enhanced p53 activity further slows down cell cycle progression and leads to cell growth inhibition. Conversely, the level of p53 is dramatically decreased upon the depletion of RPL6, indicating that RPL6 is essential for p53 stabilization. We also found that RPL6 translocalizes from the nucleolus to nucleoplasm under ribosomal stress, which facilitates its binding with HDM2. The interaction of RPL6 and HDM2 drives HDM2-mediated RPL6 polyubiquitination and proteasomal degradation. Longer treatment of actinomycin D increases RPL6 ubiquitination and destabilizes RPL6, and thereby putatively attenuates p53 response until the level of L6 subsides. Therefore, RPL6 and HDM2 form an autoregulatory feedback loop to monitor the level of p53 in response to ribosomal stress. Together, our study identifies the crucial function of RPL6 in regulating HDM2-p53 pathway, which highlights the importance of RPL6 in human genetic diseases and cancers.     

PAK1IP1, a ribosomal stress-induced nucleolar protein, regulates cell proliferation via the p53-MDM2 loop

Cell growth and proliferation are tightly controlled via the regulation of the p53-MDM2 feedback loop in response to various cellular stresses. In this study, we identified a nucleolar protein called PAK1IP1 as another regulator of this loop. PAK1IP1 was induced when cells were treated with chemicals that disturb ribosome biogenesis. Overexpression of PAK1IP1 inhibited cell proliferation by inducing p53-dependent G1 cell-cycle arrest. PAK1IP1 bound to MDM2 and inhibited its ability to ubiquitinate and to degrade p53, consequently leading to the accumulation of p53 levels. Interestingly, knockdown of PAK1IP1 in cells also inhibited cell proliferation and induced p53-dependent G1 arrest. Deficiency of PAK1IP1 increased free ribosomal protein L5 and L11 which were required for PAK1IP1 depletion-induced p53 activation. Taken together, our results reveal that PAK1IP1 is a new nucleolar protein that is crucial for rRNA processing and plays a regulatory role in cell proliferation via the p53-MDM2 loop.     

p53-Mdm2 loop controlled by a balance of its feedback strength and effective dampening using ATM and delayed feedback

When the genomic integrity of a cell is challenged, its fate is determined in part by signals conveyed by the p53 tumour suppressor protein. It was observed recently that such signals are not simple gradations of p53 concentration, but rather a counter-intuitive limit-cycle behaviour. Based on a careful mathematical interpretation of the experimental body of knowledge, we propose a model for the p53 signalling network and characterise the p53 stability and oscillatory dynamics. In our model, ATM, a protein that senses DNA damage, activates p53 by phosphorylation. In its active state, p53 has a decreased degradation rate and an enhanced transactivation of Mdm2, a gene whose protein product Mdm2 tags p53 for degradation. Thus the p53-Mdm2 system forms a negative feedback loop. However, the feedback in this loop is delayed, as the pool of Mdm2 molecules being induced by p53 at a given time will mark for degradation the pool of p53 molecules at some later time, after the Mdm2 molecules have been transcribed, exported out of the nucleus, translated and transported back into the nucleus. The analysis of our model demonstrates how this time lag combines with the ATM-controlled feedback strength and effective dampening of the negative feedback loop to produce limit-cycle oscillations. The picture that emerges is that ATM, once activated by DNA damage, makes the p53-Mdm2 oscillator undergo a supercritical Hopf bifurcation. This approach yields an improved understanding of the global dynamics and bifurcation structure of our time-delayed, negative feedback model and allows for predictions of the behaviour of the p53 system under different perturbations.     

Oscillations of the p53-Akt Network: Implications on Cell Survival and Death

Intracellular protein levels of p53 and MDM2 have been shown to oscillate in response to ionizing radiation (IR), but the physiological significance of these oscillations remains unclear. The p53-MDM2 negative feedback loop – the putative cause of the oscillations – is embedded in a network involving a mutual antagonism (or positive feedback loop) between p53 and AKT. We have shown earlier that this p53-AKT network predicts an all-or-none switching behavior between a pro-survival cellular state (low p53 and high AKT levels) and a pro-apoptotic state (high p53 and low AKT levels). Here, we show that upon exposure to IR, the p53-AKT network can also reproduce the experimentally observed p53 and MDM2 oscillations. The present work is based on the hypothesis that the physiological significance of the experimentally observed oscillations could be found in their role in regulating the switching behavior of the p53-AKT network between pro-survival and pro-apoptotic states. It is shown here that these oscillations are associated with a significant decrease in the threshold level of IR at which switching from a pro-survival to a pro-apoptotic state occurs. Moreover, oscillations in p53 protein levels induce higher levels of expression of p53-target genes compared to non-oscillatory p53, and thus influence cell-fate decisions between cell cycle arrest/DNA damage repair versus apoptosis.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G(1) arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.     

CUL4B impedes stress-induced cellular senescence by dampening a p53-reactive oxygen species positive feedback loop

Tumor suppressor p53 is known to regulate the level of intracellular reactive oxygen species (ROS). It can either alleviate oxidative stress under physiological and mildly stressed conditions or exacerbate oxidative stress under highly stressed conditions. We here report that a p53–ROS positive feedback loop drives a senescence program in normal human fibroblasts (NHFs) and this senescence-driving loop is negatively regulated by CUL4B. CUL4B, which can assemble various ubiquitin E3 ligases, was found to be downregulated in stress-induced senescent cells, but not in replicative senescent cells. We observed that p53-dependent ROS production was significantly augmented and stress-induced senescence was greatly enhanced when CUL4B was absent or depleted. Ectopic expression of CUL4B, on the other hand, blunted p53 activation, reduced ROS production, and attenuated cellular senescence in cells treated with H₂O₂. CUL4B was shown to promote p53 ubiquitination and proteosomal degradation in NHFs exposed to oxidative stress, thus dampening the p53-dependent cellular senescence. Together, our results established a critical role of CUL4B in negatively regulating the p53–ROS positive feedback loop that drives cellular senescence.     

Recurrent initiation: a mechanism for triggering p53 pulses in response to DNA damage

DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53's dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM, which is reactivated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage.     

Effect of Y220C Mutation on p53 and Its Rescue Mechanism: A Computer Chemistry Approach

Mutation causes inactivation of ‘p53’ tumor suppressor protein in almost fifty percent of cancers in humans. Outside the DNA-binding surface of p53, Y220C is the most common cancerous mutation. Previous studies have shown that a surface cavity is created by this mutation which destabilizes p53. PhiKan083, a carbazole derivative capable of binding with that cavity, and slows down its thermal denaturation rate. We investigated, theoretically, on mechanisms of structural stability loss due to Y220C mutation and mechanisms of stability restoration by PhiKan083 at the atomic level. From this study it is found that in Tp53C, Tyr220 has five electrostatic interactions with residues Val 147, Prol51, Pro153 and Pro223 located on S3/S4 loop and S7/S8 loop. The S7/S8 loop is stabilized by these electrostatic interactions. Due to the Y220C mutation all these electrostatic interactions are lost. As a result the structural fluctuation occurs at S7/S8 loop, and the loop is displaced from its original position after 6 ns MD simulation. When PhiKan083 is present (inserted) at the mutation site it provides five electrostatic interactions with Pro155, Glu221 and Thr230, and two hydrogen bonds with Leu145 and Asp228, respectively. These interactions provided by Pkikan083 stabilized the S7/S8 loop, and as a result it couldn’t be displaced. Our results showed that due to Y220C mutation p53 became destabilized through structural fluctuations surrounding the mutation site. When PhiKan083 is present at the Y220C mutation site (in 2vuk), it provides electrostatic and hydrogen bonding interactions among residue-220, its neighboring residues and PhiKan08. These interactions give additional stability to Y220C mutant p53, thus Y220C mutant p53 doesn’t destabilize.     

Pirh2, a p53-induced ubiquitin-protein ligase,promotes p53 degradation

The p53 tumor suppressor exerts anti-proliferative effects in response to various types of stress including DNA damage and abnormal proliferative signals. Tight regulation of p53 is essential for maintaining normal cell growth and this occurs primarily through posttranslational modifications of p53. Here, we describe Pirh2, a gene regulated by p53 that encodes a RING-H2 domain-containing protein with intrinsic ubiquitin-protein ligase activity. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of Mdm2. Expression of Pirh2 decreases the level of p53 protein and abrogation of endogenous Pirh2 expression increases the level of p53. Furthermore, Pirh2 represses p53 functions including p53-dependent transactivation and growth inhibition. We propose that Pirh2 is involved in the negative regulation of p53 function through physical interaction and ubiquitin-mediated proteolysis. Hence, Pirh2, like Mdm2, participates in an autoregulatory feedback loop that controls p53 function.