P53 Transactivation Research Articles

P53 Transactivation Domain Mediates Binding and Phase Separation with Poly-PR/GR

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the presence of poly-PR/GR dipeptide repeats, which are encoded by the chromosome 9 open reading frame 72 (C9orf72) gene. Recently, it was shown that poly-PR/GR alters chromatin accessibility, which results in the stabilization and enhancement of transcriptional activity of the tumor suppressor p53 in several neurodegenerative disease models. A reduction in p53 protein levels protects against poly-PR and partially against poly-GR neurotoxicity in cells. Moreover, in model organisms, a reduction of p53 protein levels protects against neurotoxicity of poly-PR. Here, we aimed to study the detailed molecular mechanisms of how p53 contributes to poly-PR/GR-mediated neurodegeneration. Using a combination of biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, fluorescence polarization, turbidity assays, and differential interference contrast (DIC) microscopy, we found that p53 physically interacts with poly-PR/GR and triggers liquid–liquid phase separation of p53. We identified the p53 transactivation domain 2 (TAD2) as the main binding site for PR25/GR25 and showed that binding of poly-PR/GR to p53 is mediated by a network of electrostatic and/or hydrophobic interactions. Our findings might help to understand the mechanistic role of p53 in poly-PR/GR-associated neurodegeneration.

HSymM-guided engineering of the immunodominant p53 transactivation domain putative peptide antigen for improved binding to its anti-p53 monoclonal antibody

A novel engineering strategy to improve autoantibody detection with peptide fragments derived from the parent antigen is presented. The model system studied was the binding of the putative p53 TAD peptide antigen (residues 46–55) to its cognate anti-p53 antibody, ab28. Each engineered peptide contained the full decapeptide epitope and differed only in the flanking regions. Since minimal structural information was available to guide the design, a simple epitope:paratope binding model was applied. The Hidden Symmetry Model, which we recently reported, was used to guide peptide design and estimate per-residue contributions to interaction free energy as a function of added C- and N-terminal flanking peptides. Twenty-four peptide constructs were designed, synthesized, and assessed for binding affinity to ab28 by surface plasmon resonance, and a subset of these peptides were evaluated in a simulated immunoassay for limit of detection. Many peptides exhibited over 200-fold enhancements in binding affinity and improved limits of detection. The epitope was reevaluated and is proposed to be the undecapeptide corresponding to residues 45–55. HSymM calculated binding free energy and experimental data were found to be in good agreement (R2 > 0.75).

The Long Non-Coding RNA SENEBLOC

Cellular senescence is a stress response and a permanent state of cell cycle arrest of normal cell division. SENEBLOC is involved in both oncogenic and replicative senescence and has been identified as a c-Myc responsive lncRNA involved in senescence. SENEBLOC acts to restrains p21-mediated senescence. Mouse double minute 2 (MDM2) regulates p53, controls its transcriptional activity and protein stability. Cyclin-dependent kinase (CDK) inhibitor p21 promotes cell cycle arrest in response to a variety of stimuli and it can be induced by both p53-dependent and p53-independent mechanisms. SENEBLOC is shown to drive both p53-dependent and p53-independent mechanisms. SENEBLOC acts as a scaffold to promote p53 turnover. It decreases p21 transactivation and promotes p53 and MDM2 association. p53-independent regulation of p21 by SENEBLOC occurs via regulatory effects on HDAC5. Rapamycin promotes SENEBLOC transcription through effects on E2F1. In this review, I focus on the importance of the newly identified LncRNA SENEBLOC

Cancer-Associated Mutations Perturb the Disordered Ensemble and Interactions of the Intrinsically Disordered p53 Transactivation Domain

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein–protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.

Development and structural characterisation of human scFv targeting MDM2 spliced variant MDM215kDa

The murine double minute 2 (MDM2) protein is a major negative regulator of the tumour suppressor protein p53. Under normal conditions, MDM2 constantly binds to p53 transactivation domain and/or ubiquinates p53 via its role as E3 ubiquitin ligase to promote p53 degradation as well as nuclear export to maintain p53 levels in cells. Meanwhile, amplification of MDM2 and appearance of MDM2 spliced variants occur in many tumours and normal tissues making it a prognostic indicator for human cancers. The mutation or deletion of p53 protein in half of human cancers inactivates its tumour suppressor activity. However, cancers with wild type p53 have its function effectively inhibited through direct interaction with MDM2 oncoprotein. Here, we described the construction of a MDM2 spliced variant (rMDM215kDa) consisting of SWIB/MDM2 domain and its central region for antibody generation. Biopanning with a human naïve scFv library generated four scFv clones specific to rMDM215kDa. Additionally, the selected scFv clones were able to bind to the recombinant full length MDM2 (rMDM2-FL). Computational prediction showed that the selected scFv clones potentially bind to exon 7–8 of MDM2 while leaving the MDM2/SWIB domain free for p53 interaction. The developed antibodies exhibit good specificity can be further investigated for downstream biomedical and research applications.

Solution-State Preorganization of Cyclic β-Hairpin Ligands Determines Binding Mechanism and Affinities for MDM2.

Understanding mechanisms of protein folding and binding is crucial to designing their molecular function. Molecular dynamics (MD) simulations and Markov state model (MSM) approaches provide a powerful way to understand complex conformational change that occurs over long time scales. Such dynamics are important for the design of therapeutic peptidomimetic ligands, whose affinity and binding mechanism are dictated by a combination of folding and binding. To examine the role of preorganization in peptide binding to protein targets, we performed massively parallel explicit-solvent MD simulations of cyclic β-hairpin ligands designed to mimic the p53 transactivation domain and competitively bind mouse double minute 2 homologue (MDM2). Disrupting the MDM2-p53 interaction is a therapeutic strategy to prevent degradation of the p53 tumor suppressor in cancer cells. MSM analysis of over 3 ms of aggregate trajectory data enabled us to build a detailed mechanistic model of coupled folding and binding of four cyclic peptides which we compare to experimental binding affinities and rates. The results show a striking relationship between the relative preorganization of each ligand in solution and its affinity for MDM2. Specifically, changes in peptide conformational populations predicted by the MSMs suggest that entropy loss upon binding is the main factor influencing affinity. The MSMs also enable detailed examination of non-native interactions which lead to misfolded states and comparison of structural ensembles with experimental NMR measurements. In contrast to an MSM study of p53 transactivation domain (TAD) binding to MDM2, MSMs of cyclic β-hairpin binding show a conformational selection mechanism. Finally, we make progress toward predicting accurate off rates of cyclic peptides using multiensemble Markov models (MEMMs) constructed from unbiased and biased simulated trajectories.

P53 mediates target gene association with nuclear speckles for amplified RNA expression

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.

Drude Polarizable Simulations of the p53 Transactivation Domain with Different Binding Partners

Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDRs) do not follow the canonical structure-function paradigm of adopting a dominant fold, instead interconverting among multiple structures that are accessible along a rugged, shallow free energy surface. This structural plasticity is exploited to facilitate binding to structurally diverse partners, including both proteins and nucleic acids. Given that IDPs often serve critical regulatory roles in a range of cellular functions such as signal transduction and cell cycle checkpoints, it is crucial to understand the dynamics and interactions of these abundant proteins. Previous molecular dynamics studies of IDPs have primarily used additive force fields, which approximate polarization effects assuming a uniform dielectric medium. This approximation may fall short in IDPs, for which cooperative, induced effects such as hydrogen bonding, are particularly important in dictating the balance of secondary structure. Here, we apply the Drude-2019 polarizable protein force field for the first time to simulations of the p53 transactivation domain (TAD) alone and in complex with two binding partners: MDM2, a protein with well-defined tertiary structure, and the nuclear coactivator domain of CBP, an IDR. p53 is a widely studied protein and is a prototypical protein with IDRs, thus the TAD is a useful model IDP to examine the role of side-chain and backbone electronic polarization in the nucleation of secondary structure upon complexation. We focus our analysis on properties that are only attainable using a polarizable force field, such as individual molecular dipole moments of different protein moieties. We find that side-chain dipole moments are perturbed upon burial in the protein-protein complexes and that these depolarization effects are almost entirely confined to the interface between the p53 TAD and the binding partners, with aliphatic residues being the most impacted.

The oncogenicity of tumor-derived mutant p53 is enhanced by the recruitment of PLK3

p53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.

Computational strategy for intrinsically disordered protein ligand design leads to the discovery of p53 transactivation domain I binding compounds that activate the p53 pathway.

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.

Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores.

Protein–protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.

A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2-DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.

MageC2 protein is upregulated by oncogenic activation of MAPK pathway and causes impairment of the p53 transactivation function

Normal-to-tumor cell transition is accompanied by changes in gene expression and signal transduction that turns the balance toward cancer-cell phenotype, eluding by different mechanisms, the response of tumor-suppressor genes. Here, we observed that MageC2, a MAGE-I protein able to regulate the p53 tumor-suppressor, is accumulated upon MEK/ERK MAPK activation. Overexpression of H-RasV12 oncogene causes an increase in MageC2 protein that is prevented by pharmacologic inhibition of MEK. Similarly, decrease in MageC2 protein levels is shown in A375 melanoma cells (which harbor B-RafV600E oncogenic mutation) treated with MEK inhibitors. MageC2 protein levels decrease when p14ARF is expressed, causing an Mdm2-independent upregulation of p53 transactivation. However, MageC2 is refractory to p14ARF-driven downregulation when H-RasV12 is co-expressed. Using MageC2 knockout A375 cells generated by CRISPR/CAS9 technology, we demonstrated the relevance of MageC2 protein in reducing p53 transcriptional activity in cells containing hyperactive MEK/ERK signaling. Furthermore, gene expression analysis performed in cancer-genomic databases, supports the correlation of reduced p53 transcriptional activity and high MageC2 expression, in melanoma cells containing Ras or B-Raf driver mutations. Data presented here suggest that MageC2 can be a functional target of the oncogenic MEK/ERK pathway to regulate p53.

A Glance of p53 Functions in Brain Development, Neural Stem Cells, and Brain Cancer.

p53 is one of the most intensively studied tumor suppressors. It transcriptionally regulates a broad range of genes to modulate a series of cellular events, including DNA damage repair, cell cycle arrest, senescence, apoptosis, ferroptosis, autophagy, and metabolic remodeling, which are fundamental for both development and cancer. This review discusses the role of p53 in brain development, neural stem cell regulation and the mechanisms of inactivating p53 in gliomas. p53 null or p53 mutant mice show female biased exencephaly, potentially due to X chromosome inactivation failure and/or hormone-related gene expression. Oxidative cellular status, increased PI3K/Akt signaling, elevated ID1, and metabolism are all implicated in p53-loss induced neurogenesis. However, p53 has also been shown to promote neuronal differentiation. In addition, p53 mutations are frequently identified in brain tumors, especially glioblastomas. Mechanisms underlying p53 inactivation in brain tumor cells include disruption of p53 protein stability, gene expression and transactivation potential as well as p53 gene loss or mutation. Loss of p53 function and gain-of-function of mutant p53 are both implicated in brain development and tumor genesis. Further understanding of the role of p53 in the brain may provide therapeutic insights for brain developmental syndromes and cancer.

P33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2

BackgroundOral squamous cell carcinoma (OSCC), a form of head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate. OSCC patients are often treated with cisplatin but resistance to chemotherapy is often observed. This makes it important identification of alternative therapeutic targets which will result in more favorable outcome in OSCC patients. The plant homeodomain (PHD)-containing protein Inhibitor of Growth family of tumor suppressor proteins (p33ING1b) has been indicated as a tumor suppressor in different cancers including OSCC. This protein has been shown to function by modulating transcriptional activity of p53; however, the exact mechanism(s) are not well defined.MethodsExpression of total and acetylated p53 and p33ING1b protein was determined in OSCC cell lines YD-9, YD-8, and YD-38 by immunoblot analysis. Effect of modulation of p33ING1b protein expression on acetylation of p53 and cell proliferation was determined by immunoblot and MTT assay. Effect of modulation of p33ING1b protein expression on transactivation of p53 was assessed by heterologous promoter-based reporter and chromatin immunoprecipitation. Effect of modulation of expression of p33ING1b on SIR2 mRNA and protein was determined by quantitative real-time PCR and immunoblot analyses. Impact of modulation of p33ING1b alone or in combination with SIR2 on chemosensitivity of YD-9 and YD-8 cells to cisplatin was determined in time and dose-dependent cell proliferation assays.ResultsHere, using a panel of OSCC cell lines with wild type or mutant p53, we show that p33ING1b expression is correlated to acetylation of p53 at lysine 382 residue. Increased acetylation of p53 following overexpression of p33ING1b was associated with increased expression of the pro-apoptotic proteins BAX, p21, and cleaved-Caspase 3, and decreased cell proliferation. Reporter assays with p21 and BAX promoters showed that p33ING1b expression levels directly correlated to promoter activity of these 2 genes. Chromatin immunoprecipitation assay showed that transcriptional regulation of p21 and BAX by acetylated p53 is dependent on expression level of p33ING1b. Differential acetylation of p53 following modulation of p33ING1b expression was indirect. Expression of p33ING1b was found to be inversely correlated to the NAD-dependent deacetylase silent information regulator 2 (SIR2). SIR2 was transcriptionally regulated by p33ING1b. Relative expression of p33ING1b was found to dictate chemosensitivity of OSCC cell lines to cisplatin treatment. Concomitant overexpression of p33ING1b and knockdown of SIR2 had a synergistic effect on chemosensitivity of OSCC cell lines to cisplatin, compared to either overexpression of p33ING1b or knockdown of SIR2 alone.ConclusionsThe results from the current study thus elucidate that p33ING1b regulates p53 acetylation irrespective of p53 mutation and subsequent transactivation by transcriptional regulation of SIR2 expression. The results also indicate that p33ING1b and SIR2 are potentially attractive therapeutic targets.

The SUMO-specific protease SENP1 deSUMOylates p53 and regulates its activity.

The stability and activity of the p53 tumor suppressor protein are tightly regulated by various posttranslational modifications, including SUMOylation. p53 can be modified by both SUMO1 and SUMO2, although how SUMOylation regulates p53 activity is still obscure. Whether p53 activity is directly regulated by deSUMOylation is also unclear. Here, we show that SENP1, a SUMO-specific protease implicated in pro-oncogenic roles, is a p53 deSUMOylating enzyme. SENP1 interacts with p53 and deSUMOylates p53 in cells and in vitro. Knockdown of SENP1 markedly induced p53 transactivation activity. We further show that SENP1 depletion synergizes with DNA damage-inducing agent etoposide to induce p53 activation and the expression of p21, leading to synergistic growth inhibition of cancer cells. Our results reveal that SENP1 is a critical p53 deSUMOylating enzyme and a promising therapeutic target in wild-type p53 containing cancer cells.

Mithramycin selectively attenuates DNA-damage-induced neuronal cell death

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates’ cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

AATF/Che-1-An RNA Binding Protein at the Nexus of DNA Damage Response and Ribosome Biogenesis.

The DNA damage response (DDR) is a complex signaling network that is activated upon genotoxic stress. It determines cellular fate by either activating cell cycle arrest or initiating apoptosis and thereby ensures genomic stability. The Apoptosis Antagonizing Transcription Factor (AATF/Che-1), an RNA polymerase II-interacting transcription factor and known downstream target of major DDR kinases, affects DDR signaling by inhibiting p53-mediated transcription of pro-apoptotic genes and promoting cell cycle arrest through various pathways instead. Specifically, AATF was shown to inhibit p53 expression at the transcriptional level and repress its pro-apoptotic activity by direct binding to p53 protein and transactivation of anti-apoptotic genes. Solid and hematological tumors of various organs exploit this function by overexpressing AATF. Both copy number gains and high expression levels of AATF were associated with worse prognosis or relapse of malignant tumors. Recently, a number of studies have enabled insights into the molecular mechanisms by which AATF affects both DDR and proliferation. AATF was found to directly localize to sites of DNA damage upon laser ablation and interact with DNA repair proteins. In addition, depletion of AATF resulted in increased DNA damage and decrease of both proliferative activity and genotoxic tolerance. Interestingly, considering the role of ribosomal stress in the regulation of p53, more recent work established AATF as ribosomal RNA binding protein and enabled insights into its role as an important factor for rRNA processing and ribosome biogenesis. This Mini Review summarizes recent findings on AATF and its important role in the DDR, malignancy, and ribosome biogenesis.

Selective degradation of IKK\u03b1 by autophagy is essential for arsenite-induced cancer cell apoptosis

Two catalytic subunits of the IKK complex, IKKα and IKKβ, trigger NF-κB activation as well as NF-κB-independent signaling events under both physiological and pathological conditions. Here we identified the NF-κB-unrelated cytoprotective function of IKKα in promoting autophagy by triggering p53 transactivation and upregulation of its downstream autophagic mediator, DRAM1, in the arsenite-treated hepatoma cells, which responses depended on IKKα kinase activity. Furthermore, IKKα triggered p53/DRAM1-dependent autophagy by inducing CHK1 activation and CHK1/p53 interaction. Interestingly, after provoking autophagy, IKKα could be specifically recognized by the autophagic machinery via directly binding with LC3B, resulting in selective degradation of IKKα by autophagy. Unexpectedly, the selectivity of autophagic sequestration towards IKKα was mediated by novel mechanism independent of the classical LC3-interacting regions (LIRs) within IKKα, while C-terminal arm of LIR was involved in mediating IKKα/LC3B interaction. Taken together, we conclude that IKKα attenuates arsenite-induced apoptosis by inducing p53-dependent autophagy, and then selective feedback degradation of IKKα by autophagy contributes to the cytotoxic response induced by arsenite.

Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells.

Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway.