Water's potential role: Insights from studies of the p53 core domain

Soluble proteins with amyloidogenic propensity such as the tumor suppressor protein p53 have high proportion of incompletely desolvated backbone H bonds (HB). Such bonds are vulnerable to water attack, thus potentially leading to the misfolding of these proteins. However, it is still not clear how the surrounding solvent influences the protein native states. To address this, systematic surveys by molecular dynamics simulations and entropy analysis were performed on the p53 core domain in this work. We examined seven wild/mutant X-ray structures and observed two types of water-network hydration in three "hot hydration centers" (DNA- or small molecule- binding surfaces of the p53 core domain). The "tight" water, resulting from the local collective hydrogen-bond interactions, is probably fundamental to the protein structural stability. The second type of water is highly "dynamical" and exchanges very fast within the bulk solution, which is unambiguously assisted by the local protein motions. An entropy mapping of the solvent around the protein and a temperature perturbation analysis further present the main features of the p53 hydration network. The particular environment created by different water molecules around the p53 core domain also partly explains the structural vulnerabilities of this protein.     

Oligomerization of p53 Precedes its Association with Dynein and Nuclear Accumulation

Previous studies have identified several proteins that associate with microtubules and the dynein motor complex including p53, the glucocorticoid and the vitamin D receptors, and the APC (adenomatous polyposis coli) protein; but neither the residues important for this interaction nor the physical state of the proteins involved have been clarified. We observed in SN12C cells harboring a mutant p53 truncated at amino acid 336, impaired nuclear localization and impaired association with dynein. This finding was confirmed and extended by examining a series of truncated p53 proteins that identified residues 336 to 348 as crucial for association with dynein and nuclear transport. Point mutations identified the importance of residues involved in p53 oligomerization in this process, establishing a p53 oligomer as the cargo for dynein transport. The association of cytosolic p53 oligomers with dynein occurs independent of microtubules indicating that following this association, the p53/dynein complex then associates with microtubules and is transported to the peri-nuclear region. These studies suggest that mutations or modifications that affect p53 oligomerization not only interfere with DNA binding but also with its intracellular distribution. They also highlight the importance of an intact microtubule network in the trafficking of crucial cellular proteins.     

The coordinated interaction of multifunctional members of p53 family determines many key processes in multicellular organisms

First time p53 was found in the complex with viral large T-antigene in the cells transformed by small DNA virus SV40. The cloning of p53 cDNA was done in the beginning of eighties and soon after that the whole p53 gene was cloned. The p53 family is comprised of three genes: TP53,TP63 and TP73, each of which is expressed as a set of structurally and functionally different isoforms. All of them intensively interact with each other forming a united functional network of proteins. In this review we discuss evolution of the p53 family and significance of all its members in embryonic development, reproduction, regeneration, regulation of aging and life span, as well as in the body's defense against cancer. With special attention we review the role of less studied members of the p53 family: p63 and p73, in oncogenesis and tumor progression and show that different isoforms of these proteins might exert a contrary effect on these processes.     

P53 licensed to kill? Operating the assassin

The p53 protein is a key player in the cellular response to stress. Proper regulation of p53 is imperative for the suppression of tumor development. This regulation is largely governed by its master inhibitor, Mdm2, which both blocks p53 activities and promotes its destabilization. This tight regulation of p53 by Mdm2 must be interrupted under stress conditions in order for p53 to be stabilized in an active form. A combined action of partner proteins and modifying enzymes is essential for the relief of p53 from Mdm2. The recent revelation of p53 association with the PML-nuclear bodies provides one explanation of how this regulatory network is coordinated within the nucleus in response to certain stress conditions. Thus, it is not only the nature of the p53 regulatory complex but also the spatial and temporal context of this association that governs the output inhibitory signals mediated by p53.     

Coordinated interaction of multifunctional members of the p53 family determines many key processes in multicellular organisms

For the first time, p53 was found in complex with the viral large T-antigen in cells transformed with the small DNA virus SV40. p53 cDNA was cloned in the early 1980s, and the full-length p53 gene was cloned soon afterwards. The p53 family is comprised of three genes—TP53, TP63, and TP73—each of which is expressed as a set of structurally and functionally different isoforms. All of them intensely interact with each other, forming a united functional network of proteins. The review discusses the evolution of the p53 family and the significance of all its members in embryo development, reproduction, regeneration, regulation of aging and lifespan, and defense against cancer. Special attention is paid to the role of poorly studied members of the p53 family, p63 and p73, in carcinogenesis and tumor progression. Different isoforms of these proteins might exert opposite effects on these processes.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

Coordination between p21 and DDB2 in the Cellular Response to UV Radiation

The tumor suppressor p53 guides the cellular response to DNA damage mainly by regulating expression of target genes. The cyclin-dependent kinase inhibitor p21, which is induced by p53, can both arrest the cell cycle and inhibit apoptosis. Interestingly, p53-inducible DDB2 (damaged-DNA binding protein 2) promotes apoptosis by mediating p21 degradation after ultraviolet (UV)-induced DNA damage. Here, we developed an integrated model of the p53 network to explore how the UV-irradiated cell makes a decision between survival and death and how the activities of p21 and DDB2 are modulated. By numerical simulations, we found that p53 is activated progressively and the promoter selectivity of p53 depends on its concentration. For minor DNA damage, p53 settles at an intermediate level. p21 is induced by p53 to arrest the cell cycle via inhibiting E2F1 activity, allowing for DNA repair. The proapoptotic genes are expressed at low levels. For severe DNA damage, p53 undergoes a two-phase behavior and accumulates to high levels in the second phase. Consequently, those proapoptotic proteins accumulate remarkably. Bax activates the release of cytochrome c, while DDB2 promotes the degradation of p21, which leads to activation of E2F1 and induction of Apaf-1. Finally, the caspase cascade is activated to trigger apoptosis. We revealed that the downregulation of p21 is necessary for apoptosis induction and PTEN promotes apoptosis by amplifying p53 activation. This work demonstrates that how the dynamics of the p53 network can be finely regulated through feed-forward and feedback loops within the network and emphasizes the importance of p21 regulation in the DNA damage response.     

Surface-enhanced Raman scattering detection of wild-type and mutant p53 proteins at very low concentration in human serum

The development of ultrasensitive and rapid approaches to detect tumor markers at very low concentrations even in a physiological environment represents a challenge in nano-medicine. The p53 protein is at the center of the cellular network that protects organisms against the insurgence of tumors, most of which are related to alteration of p53 expression. Therefore p53 is regarded as a valuable prognostic marker whose detection at high sensitivity may considerably contribute to early diagnosis of cancers. In this work we have applied an analytical method based on surface enhanced Raman spectroscopy with high sensitivity and rapidity to improve traditional bioaffinity techniques. The Raman reporter bifunctional linker 4-aminothiophenol (4-ATP) first assembled onto 50 nm gold nanoparticles (Nps) has then been azotated to bind low concentration wild-type and two mutated forms of p53 proteins. The Raman signal enhancement of the resulting p53-(4-ATP-Np) systems has been used to identify the p53 molecules captured on a recognition substrate constituted by the azurin (Az) protein monolayer. Az has shown a strong association for both wild-type and mutated p53 proteins, allowing us to selectively detect these proteins at concentrations as low as 500 fM, in a human serum environment.     

The p53-MDM2 network: from oscillations to apoptosis

The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective.We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.     

The p53-MDM2 network: from oscillations to apoptosis

The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.     

Adaptive homeostasis and the p53 isoform network

All living organisms have developed processes to sense and address environmental changes to maintain a stable internal state (homeostasis). When activated, the p53 tumour suppressor maintains cell and organ integrity and functions in response to homeostasis disruptors (stresses) such as infection, metabolic alterations and cellular damage. Thus, p53 plays a fundamental physiological role in maintaining organismal homeostasis. The TP53 gene encodes a network of proteins (p53 isoforms) with similar and distinct biochemical functions. The p53 network carries out multiple biological activities enabling cooperation between individual cells required for long‐term survival of multicellular organisms (animals) in response to an ever‐changing environment caused by mutation, infection, metabolic alteration or damage. In this review, we suggest that the p53 network has evolved as an adaptive response to pathogen infections and other environmental selection pressures.     

Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors

p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha). The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications. Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors. Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins. The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable. Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins. Having several features in common with p53, the p51 proteins may function in biological processes apart from p53.     

The Dynamics of Stress p53-Mdm2 Network Regulated by p300 and HDAC1

We construct a stress p53-Mdm2-p300-HDAC1 regulatory network that is activated and stabilised by two regulatory proteins, p300 and HDAC1. Different activation levels of observed due to these regulators during stress condition have been investigated using a deterministic as well as a stochastic approach to understand how the cell responds during stress conditions. We found that these regulators help in adjusting p53 to different conditions as identified by various oscillatory states, namely fixed point oscillations, damped oscillations and sustain oscillations. On assessing the impact of p300 on p53-Mdm2 network we identified three states: first stabilised or normal condition where the impact of p300 is negligible, second an interim region where p53 is activated due to interaction between p53 and p300, and finally the third regime where excess of p300 leads to cell stress condition. Similarly evaluation of HDAC1 on our model led to identification of the above three distinct states. Also we observe that noise in stochastic cellular system helps to reach each oscillatory state quicker than those in deterministic case. The constructed model validated different experimental findings qualitatively.